Name: Homarus Americanus Stomatogastric Nervous System Dissection (Video) | JoVE Journal
Uploaded: 2009-05-28T00:00:00.000+00:00
Duration: 26 min 22 s
Description: Learn fine dissection of the stomatogastric nervous system from Homarus americanus for neuroscience research. This protocol isolates ganglia and nerves to study neuronal function in rhythmic behaviors.

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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/pubmed?term=((Central+pattern+generators+and+the+control+of+rhythmic+movements%5BTitle%5D)+AND+%22Curr.+Biol%22%5BJournal%5D)">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/pubmed?term=((A+small-systems+approach+to+motor+pattern+generation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((In+vivo+modulation+of+interacting+central+pattern+generators+in+lobster+stomatogastric+ganglion%3A+influence+of+feeding+and+partial+pressure+of+oxygen%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Gross+dissection+of+the+stomach+of+the+lobster%2C+Homarus+americanus%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((The+structure+of+the+stomatogastric+neuromuscular+system+in+Callinectes+sapidus%2C+Homarus+americanus+and+Panulirus+argus+%28Decapoda+crustacean%29%5BTitle%5D)+AND+%22Philos.+Trans.+R.+Soc.+Lond.+B+Biol.+Sci%22%5BJournal%5D)">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/pubmed?term=((Natural+history+of+the+American+lobster%5BTitle%5D)+AND+%22Bull.+U.S.+Bur.+Fish%22%5BJournal%5D)">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/pubmed?term=((Understanding+circuit+dynamics+using+the+stomatogastric+nervous+system+of+lobsters+and+crabs%5BTitle%5D)+AND+%22Annu.+Rev.+Physiol%22%5BJournal%5D)">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/pubmed?term=((Central+pattern+generating+neurons+simultaneously+express+fast+and+slow+rhythmic+activities+in+the+stomatogastric+ganglion%5BTitle%5D)+AND+%22J.+Neurophysiol%22%5BJournal%5D)">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., Fénelon, V., Baro, D. J., Harris-Warrick, R. M., Meyrand, P., Simmers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+maintenance+of+channel+distribution+in+a+central+pattern+generator+neuron+by+neuromodulatory+inputs+revealed+by+decentralization+in+organ+culture%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Variable+channel+expression+in+identified+single+and+electrically+coupled+neurons+in+different+animals%5BTitle%5D)+AND+%22Nat.+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Quantitative+expression+profiling+of+identified+neurons+reveals+cell-specific+constraints+on+highly+variable+levels+of+gene+expression%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">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.

<table><tbody><tr><td>Fine Tungsten Wire (0.001 in. diameter)</td><td>Tool</td><td>California Fine Wire Company</td><td></td><td>Cut into 1-2mm pieces to use as fine pins.</td></tr><tr><td>Forceps #5</td><td>Tool</td><td>Fine Science Tools</td><td>91150-20</td><td></td></tr><tr><td>Minuten Pins</td><td>Tool</td><td>Fine Science Tools</td><td>26002-10</td><td></td></tr><tr><td>Moria Spring Scissors (7mm blades)</td><td>Tool</td><td>Fine Science Tools</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>Fisher Scientific</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></td></tr><tr><td>Pin Holder</td><td>Tool</td><td>Fine Science Tools</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>Fine Science Tools</td><td>11252-00</td><td></td></tr><tr><td>Tungsten Needles</td><td>Tool</td><td>Fine Science Tools</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></td><td></td><td></td><td></td></tr></tbody></table>

homarus americanus stomatogastric nervous system dissection

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.

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 ...

The procedure for isolating the ST gastric nervous system from the lobster can be divided into two parts, the gross and the fine dissection. This video covers the fine dissection of the somatic gastric nervous system from the stomach, including the steato gastric ganglion referred to as the STG and the commissural ganglia referred to as the cogs. Essentially, the nerves and ganglia must be separated from the surrounding tissue, beginning with isolation of the cgs, followed by isolation of the St G.The result of this procedure is the systematic gastric nervous system pinned out in a dish ready to be used for a variety of studies focused on nervous system function.Hi, I'm Annalise Tobin from the laboratory of Dr.Eve Martyr in the biology department at Brandeis University. Hi, I'm Hillary Burman, also from the Martyr Lab. Today we will show you a protocol for isolating the somatic gastric nervous system from the stomach of the lobster homarus Americana to see how the stomach was removed.Please see our accompanying video protocol in JoVE. We use this procedure in our laboratory to study the function of neurons in small neuro circuits in producing rhythmic behaviors. So let's get started.To begin, a preparation of the lobster stomach is required to see how this is accomplished. See the accompanying video by a beerman and Tobin first orient the dish so that the interior end, which is by the lip, is away from you and the posterior end is toward you. This part of the procedure begins with a more precise pinning of the preparation.Pin down the esophagus on each side of the lip, and if necessary, rearrange the other pins placed during the gross dissection so the stomach lays flat. And next, remove the hypodermis by first grabbing it near the gm, two GM three muscles, detaching it from these muscles and gently pulling it in the anterior direction. Separate the hypodermis from the underlying fat by snipping any connections using grease care around the central artery, which encloses part of the somatic gastric nervous system leave the hypodermis intact.Within about a 0.5 centimeter radius of the somatic gastric ganglion, which sits between the GM one B muscles, the separated hypodermis can now be cut off. The next step is to detach the brain from the underlying tissue. Begin by identifying the two large commis nerves that project bilaterally from the anterior portion of the brain to the Commis ganglia in the anterior portion of the stomach.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 somatic gastric nerve is underneath the brain and must be kept intact. To separate the commissural ganglia from the surrounding tissue, first locate them by following one of the comm nerves to a ganglion.Now identify the ION and SON. These nerves must remain intact. Each commis ganglion will have many nerves radiating out.Cut most of them except the ION and SON. And leave one extra nerve attached to hold the commis ganglion in place while you finish the dissection. Now locate and trim the nerves from the other commis ganglion in the same manner.Next, clean the ION and SON from lateral to medial. Leave the labial nerve intact to anchor the prep to the stomach. The esophageal ganglion sits with the iOS intersect.You can clear below it by grabbing the inferior ventricular nerve and pointed upwards to cut beneath. The next step is to separate the STG from the surrounding tissue. The STG sits inside an artery that runs between the GM one B muscles and down to the GM two a B muscles.Look for the cut end of the artery near the gm. Two AB muscle pull the artery up and interiorly to expose the DVN. Continue pulling up to the remaining hypodermis.Once separated, the artery can be trimmed to the level where it touches the remaining hypodermis. Now cut off the muscle flap that was situated in the rostrum of the lobster. Next, lift up the hypodermis above the artery.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. Clear any tissue away from the STN. Now we can proceed to isolate several other nerves.The next nerves to separate from the surrounding tissue are the AGNs. Even if you do not plan to record from the A GN, you will need a portion of it to pin down the ganglion. To better see the AGNs, remove the remaining hypodermis and a thin layer of the underlying muscle just posterior to the STG.Look for a dark tunnel in the fat and muscle on either side of the DVN 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 five millimeters of the A GN if you plan to record from it.Detach the a GN from the muscle it innervates by cutting the connections. And now repeat this process on the other side to isolate the other A GN.Once both AGNs have been dissected, cut all the muscles and tissue connecting the STG to the stomach. Next, isolate the M vns.They are a bilateral pair of small nerves branching off the LVN and traversing laterally underneath the fat. 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 M VNS and won't risk cutting them.Lift up the fat layer and look underneath for a small nerve. The M VNS run media laterally just posterior to the level of the GM one B muscles. 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 and any attachments to the M VNS at the end of the nervous system dissection. Repeat the process on the other side to isolate the other MVN. Next in line for isolating are the LVNs in the posterior nerves.Starting with 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.Continue revealing the LVN until the point where the PSN branches off. Now locate the PYN. It is a nerve traveling posteriorly over the pori.Remove the thin membrane covering this region. Isolate the PYN by cutting a small section of the pori attached to the PYN can. Next gently lift the pric section and cut away the tissue on either side of the PYN until you reach the branch that attaches to the PDN.Proceed with isolating the PDNA nerve traveling posteriorly to the pric dilator muscle. First cut a small section of the pric dilator muscle attached to the PDN. Then gently lifting the pric dilator muscle section.Cut away the tissue on either side of the PDN until you reach the branch that attaches to the PYN. Now locate the ventral LVN that connects the PYN and PDN to the LVN 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 lifting on the handle.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. You have now successfully dissected the posterior nerves on one side of the stomach. Now isolate the LVN and the posterior nerves on the other side.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.Next, cut the lateral end of each MVN. Lift the lateral end and cut any attachments to the MVN at the anterior end. Cut any attachments to the comm ganglia and cut the labial nerve on each side.Visually scan the somatic gastric nervous system to identify and then cut any additional attachments to the stomach tissue. And once you have cut all connections, you will be able to grab onto the anterior end of both commis nerves above the Commis ganglia and peel the somatic gastric nervous system off the stomach and move it to the side of the dish. As you do this, cut any additional attachments to free the nervous system.The dissection is now complete. Begin by preparing the sogar dish to which the nervous system will be pinned. Rub some stomach tissue thoroughly on the surface of the sogar in the small dish.This will decrease the hydrophobicity of the sogar so that saline will distribute evenly across the dish. Rinse and fill the sigar dish with chilled sailing. Transfer the nervous system to the prepared sogar dish by grabbing the anterior ends of both commis nerves.Lifting the nervous system out of the large dish and laying it in the small dish. To pin down the nervous system, use a combination of minutia pins and thinner pins. Cut from tungsten wire first, ensure that the preparation is dorsal side up by checking that the inferior ventricular nerve is pointing away from the syl guard.Now remove the brain and pin down the commis nerves. Gently pull the nervous system tied and flat by pulling each of the PSN posteriorly and laterally and pinning them down. Next, remove the P dilator muscle from the PNS and pin down those nerves.Then remove the pyloric segment from the GYNs and pin down the GYNs. Lastly, pin down any remaining isolated nerves such as the labial nerves, the m vns, and the AGNs. The next step is to de sheet the S st.G.First, ensure that the S st G is flat against the S guard and held tau by the lateral AGNs. The anterior STN and the posterior DVN arrange the lighting to enable visualization of the axons within the DVN. Then using a small pin or fine tungsten needle.Make a small shallow hole in sheath surrounding the DVN, just posterior to the STG. Pull the needle across the DVN stained dorsal to the axons to create a horizontal tear in the nerve sheath. Now grab a flap of nerve sheath posterior to the STG and pull it anteriorly and cut to remove the sheath from the dorsal side of the STG.Once the sheath is removed, the preparation is ready for experimentation provided the preparation is kept cool between approximately four and 15 degrees Celsius. This preparation has been noted to stay experimentally stable for as long as five days. The intact Sato gastric nervous system should be pin T against the Syl guard dish.As shown the nerves should be free of damage and accessible for extracellular recordings. The cell bodies of the intact STG should be visible and accessible for intracellular recordings. We've just shown you how to isolate the somatic gastric nervous system of the lobster Homa Americana.When doing this dissection, it's important to become very familiar with the anatomy to avoid damaging any tissue and nerves. Also, it's critical to keep the preparation at a cool temperature anywhere between four to 15 degrees Celsius. This can easily be achieved during the dissection by changing the saline out every half an hour with cool saline that you keep in the refrigerator at four degrees Celsius when the preparation is kept cool.It's viable for hours up to days. So that's it. Thank you for watching and good luck with your experiments.

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Research

JoVE Journal

Biology

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Cancer Borealis Stomatogastric Nervous System Dissection

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Gross Dissection of the Stomach of the Lobster, Homarus Americanus

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Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

Here we present a protocol for minimally invasive surgical lesioning of muscles intrinsic to the feeding apparatus of the marine mollusk Aplysia californica to understand the roles of these muscles during feeding behavior.

A Minimally Invasive Lesion Technique for Muscles Intrinsic to the Odontophore of Aplysia californica

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

Learn fine dissection of the stomatogastric nervous system from Homarus americanus for neuroscience research. This protocol isolates ganglia and nerves to study neuronal function in rhythmic behaviors.

Homarus Americanus Stomatogastric Nervous System Dissection

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