We gratefully acknowledge the members of the Marder Lab. We thank our advisor, Dr. Eve Marder, for her support and encouragement. This research was supported by the National Institute of Neurological Disorders and Stroke Grants NS-17813 and NS-058110.

1. Gross Dissection
<ol>
	<li>Place a crab on ice for 30 minutes to dull the sensory and motor systems of the animal. Surround it on all sides with ice. Gloves should be worn when handling the crab.</li>
	<li>Prepare for the gross dissection by laying out the dissection pan and the tools you will use. Rongeurs, bone-cutting scissors, small scissors, toothed forceps, a spatula with a tapered edge, a black Sylgard-coated dish, and insect pins are needed.</li>
	<li>Remove the crab from the ice bucket by its hind leg to prevent it from pinching and place it in the dissection pan.</li>
	<li>Remove the claws first by twisting each one inward. Then remove each leg using the same twisting motion.</li>
	<li>Using the rongeurs, remove the maxilla covering the mouth. Also remove the soft maxillule.</li>
	<li>Using the rongeurs again, start from the lateral posterior end of the carapace and get a good grip on the edge of it. Clamp down with the rongeurs and break that piece of carapace off. Make sure to remove enough of the carapace to allow the spatula to enter the opening. Continue removing the edge of the carapace up to the eyes and repeat on the other side.</li>
	<li>With the tapered side of the spatula, gently scrape the soft inner tissue away from the roof of the dorsal carapace. Keep the spatula in contact with the carapace to avoid damaging the STNS. Use the rongeurs to break away the parts of the carapace that have been separated from the tissue. Also separate some of the soft tissue from the ventral carapace making sure to separate any adductor muscles attaching the tissue to the carapace.</li>
	<li>Break away the dorsal carapace as far posterior as the heart where there are two small ossicles protruding from the carapace. Cut as far anterior as possible. Stabilize the cephalon (the &#8220;face&#8221;) with one finger while breaking away the most anterior parts of the dorsal carapace.</li>
	<li>On the ventral side of the carapace, pull out the mandibles one at a time by anchoring the rongeurs onto a mandible, twisting them outwards to dislodge the mandible from the attached epistome, and pulling it away. Two long ossicles with muscles should be attached to the extracted mandible if done properly.</li>
	<li>Separate away the tissue from the sides of the cephalon near the eyes using the spatula. With small dissection scissors, cut across the tissue as near to the ridge of ossicle as possible layer by layer.</li>
	<li>Rest the crab against a wall of the dissection pan and prop it up securing it in place (the claws may be used for this). Cut the labrum (&#8220;upper lip&#8221;) away from the epistome making sure not to leave any tissue attached. Using bone-cutting scissors make two diagonal cuts into the ventral carapace to remove the cephalon.</li>
	<li>Take the labrum with toothed forceps and cut the labium (&#8220;lower lip&#8221;) away from the ossicle on the ventral side. Then gently lift the lip away from the remaining ventral carapace. Use the small scissors to separate the ventral part of the stomach from the tissue lining the carapace. When the pyloric ampullae are visible, cut the stomach away from the tissue on either side of it until the stomach is extracted from the crab.</li>
	<li>Still holding the stomach by the lip with forceps, rest the stomach against the palm of the hand and pour saline into the mouth. This expands the stomach and makes it easier to cut open. Cut from the opening at the mouth down between the pylorus right through the ossicle between the ampullae. Then cut diagonally down through the two cardial branches. Cut off the tips of the three teeth inside of the stomach.</li>
	<li>Place the stomach onto a large, black, Sylgard-coated dish with the inside of the stomach down. Pin down the five corners of the preparation with insect pins and fill the dish with cold saline, immersing the entire preparation (see figure 1).</li>
</ol>
2. Fine Dissection
<ol>
	<li>, Micro-dissection tools are needed for the fine dissection. In particular, forceps and spring scissors are needed as well as a pin holder with a tungsten needle. A clear, Sylgard-coated, petri dish and some fine wire pins should also have been prepared.</li>
	<li>Beginning on the lowest magnification, start by pinning down the flaps near the lip that used to be the mouth opening. Rearrange the rest of the pins to make the preparation taut and pin any other areas necessary.</li>
	<li>Carefully lift the spotted hypodermis covering the lower part of the preparation and trim it across leaving a patch of hypodermis with two white dots.</li>
	<li>Peel up the yellow, wispy, membranous tissue near the bottom of the preparation. The mvns are weakly attached to the underside of this tissue. Carefully separate the mvns from the yellow tissue as it is pulled up. Follow the yellow tissue up and cut alongside the brain.</li>
	<li>Follow the thick processes exiting the brain that extend laterally. These are the commisural nerves. Once the end of the commissural nerve is reached, remove the mass of spongy, off-white tissue that has been cut away from the side of the brain.</li>
	<li>Where the ions and sons meet the CoGs, cut away the muscle and tissue to reveal the length of the ions and sons.</li>
	<li>Return to the spotted hypodermis patch and cut it off. This should leave an opening in the artery surrounding the STG. Cut through the opening to separate the two muscles that flank the STG. Lift the ends of these muscles and cut right underneath them to separate them from the artery and to expose the alns and agns. Separate the flanking muscles as far up as the cartilage lying over the stn.</li>
	<li>Cut away the arterial tissue surrounding the STG down to where the mvns, dgn, and dvn meet. Work down the dvn to the lvns and cut through the whitish, delicate tissue covering the lvns.</li>
	<li>Locate the psn over the ossicle near the bottom of the preparation. The psn can be used to locate the lvn and dlvn junction.</li>
	<li>Next follow the dlvn down to locate the pyn and pdn. The pyn often wraps over the pyloric ampulla to the opening. The pdn forks off of the dlvn and is between the pyloric ampulla and the cardio pyloric valve muscles. Leave some muscle attached to one of these nerves because they can be easily confused when the preparation is transferred to the clear Sylgard dish.</li>
	<li>Once all of the nerves have been uncovered, sever any remaining connections between the STNS and the tissue and unwanted nerves of the stomach. Carefully move the STNS away from the rest of the stomach, cutting any missed connections.</li>
	<li>Condition the clear Sylgard dish with the remaining mass of stomach tissue by rubbing it over the surface of the Sylgard until the Sylgard no longer feels too sticky or dry. Sylgard is hydrophobic and the STNS will adhere strongly to it if the Sylgard-coated dish is not conditioned in this manner.</li>
	<li>Add some cold saline to the dish. Grab the upper ends of the commissural nerves with forceps and bring the STNS to the clear Sylgard dish.</li>
	<li>Pin the STNS down onto the Sylgard. Sever the brain from the commissural nerves and use the minuten pins to secure the four ends of the CoGs down first. Ensure that the preparation is right side up by checking that the ivn is pointing up away from the Sylgard. The dgn should exit slightly beneath the STG when the STG is right-side up. Pin the rest of the nerve ends down with the fine wire pins.</li>
	<li>Clean away any remaining muscle or tissue from the STNS.</li>
	<li>Desheath the STG using a pin holder and a slightly hooked, fine, tungsten needle. Make a small hole in a corner of the STG sheath away from the cell bodies and use that hole to get between the layers of sheath. Carefully cut a flap of separated sheath to expose the STG neuropil and cell bodies.</li>
	<li>Pin any flaps of sheath down to increase accessibility to the cell bodies and to stabilize the STG for intracellular recordings. Re-pin the rest of the STNS ensuring that each nerve is taut and well spaced for extracellular recording (see figure 3).</li>
</ol>
3. Results
<ol>
	<li>Ideally, all of the nerves should be free of nicks and damage, particularly the ones that will be recorded from. None of the nerves should be tangled or twisted. The STG should be intact with all of the cells arranged in a beard formation around the neuropil. The intact STNS is bilaterally symmetrical and looks like a homunculus with the lvns as the legs, the mvns as the arms and the anterior end as the head.</li>
</ol>
Abbreviations:
<table border="1">
	<tbody>
		<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>OG</td>
			<td>Oesophageal ganglion</td>
		</tr>
		<tr>
			<td>mvn</td>
			<td>Median 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>aln</td>
			<td>Anterior lateral nerve</td>
		</tr>
		<tr>
			<td>dgn</td>
			<td>Dorsal 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>pyn</td>
			<td>Pyloric nerve</td>
		</tr>
		<tr>
			<td>pdn</td>
			<td>Pyloric dilator nerve</td>
		</tr>
		<tr>
			<td>dlvn</td>
			<td>Dorsal branch of the lateral ventricular nerve</td>
		</tr>
		<tr>
			<td>ivn</td>
			<td>Inferior ventricular nerve</td>
		</tr>
	</tbody>
</table>
<img alt="Figure 1" src="/files/ftp_upload/1207/1207fig1.jpg" />
Figure 1: Diagram illustrating the approximate placement of the STNS in the stomach prior to dissection.
<img alt="Figure 2" src="/files/ftp_upload/1207/1207fig2.jpg" />
Figure 2: Diagram of the various muscles in the lower part of the stomach and an overlay of the STNS in light green. Each cell type in the STG is listed with the muscles they are known to innervate. This map is helpful when planning to dissect a nerve which carries a signal from a given cell (courtesy of the Nadim lab).
<img alt="Figure 3" src="/files/ftp_upload/old/1207_2009_1_12_5/figure_3.jpg" /> 
Figure 3: Schematic diagram of the STNS with intracellular electrodes and extracellular wells (from Marder and 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>

The STNS dissection is the first step in performing an electrophysiology or immunohistochemistry experiment, or for obtaining cells for cell culture. Regardless of the intended experiment, the gross dissection will remain unchanged. However, the fine dissection may vary. It is important to plan ahead of time which cells and which nerves you will record from. Most neurons in the STG have processes that innervate at least one muscle in the stomach (see figure 2) and are classified according to their innervated target. The...

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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

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.

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

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.

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 ...

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12.5K Görüntüleme.  Brandeis. Jonah yengeç stomatogastric sinir sistemi (STNS) ( C. poyraz Elektrofizyoloji, immünhistokimya ve hücre kültürü çalışmaları için kullanılabilir). STNS çıkarma iki bölümden yapılır: kaba ve ince diseksiyon.

Video: Cancer Borealis 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 ...

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).

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.

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

A BW Reporter System for Studying Receptor-Ligand Interactions

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native receptor and the ligand are challenging since the ligand of a specific receptor may be unknown and experimental procedures with the native ligand can be technically complicated. To address these obstacles, we describe a reporter system to detect the binding and activation of a specific receptor by a ligand of interest. In this reporter system, the extracellular domain of a specific receptor is conjugated to mouse CD3&#950; and this chimeric protein is then expressed in mouse BW cells. These transfected BW cells can then be incubated with different targets (e.g., cells or antibodies). Activation of a transfected receptor leads to the secretion of mouse interleukin-2 (mIL-2) which can be detected by enzyme-linked immunosorbent assay (ELISA). This reporter system has the advantages of being sensitive and specific to a single receptor. In addition, the activation level of a specific receptor can easily be quantified and can be used even in cases where the ligand of the receptor is unknown. This system has been implemented successfully in many of our studies to characterize receptor-ligand interactions. We have recently employed this system to study the activation of human Fc&#947; receptors (Fc&#947;Rs) by different monoclonal anti-CD20 antibodies in clinical use.

This protocol describes how to establish a reporter system that can be used to identify and quantify receptor-ligand interactions.

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...

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 ...