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.
