The procedure for isolating the stemo gastric nervous system from a lobster can be divided into two parts, the gross and the fine dissections. In this video, we demonstrate the gross dissection. The protocol involves isolating the cephalic thax, removing the stomach, and opening the stomach to facilitate the fine dissection of the somatic gastric nervous system.
Hi, I am Hilary Bierman from the laboratory of Dr.Eve Martyr at Brenda University. Hi, I'm Annalise Tobin, also from the murder lab. Today we will show you the procedure for the gross dissection of the stomach of the lobster Hamas Americana.
We use this procedure in our laboratory to access the somatic gastric nervous system, which we used to study how small neural circuits produce rhythmic behavior. So let's get started. 30 minutes before the dissection anesthetize the lobster by burying it in ice at the beginning of a dissection.
The sex of a lobster is determined by looking at the shape of the first swim ette. Males have bony swim ettes and females have feathery swim ettes. Begin the dissection by first isolating the cephalic thax.
The first step in doing this is applying a twisting motion to rip off the front claws. Next, remove the abdomen. Begin at the tail portion by using the large scissors and make a deep cut between the celo, thax and the abdomen.
This cuts the giant fiber tracks controlling the tail flip response. And now finish cutting off the abdomen. Remove the antenna by trimming them off with scissors and twist off the legs and the third maxillipeds.
And next, you will reveal the stomach within the clu thax by removing the surrounding carpus. The stomach is between the cervical groove and the rostrum. Starting on the lateral edge of this section, user on Jos and spatula.
To carefully remove the hard carus while leaving the reddish underlying hypodermis intact. The spatula is useful for separating the layers so that the carpus can be broken off. With the Ron Jos moving toward the eyes, you find bilateral, lightly calcified small oval plates.
These are the three A OSLs as close as possible. To these ossicles, carefully detach the muscle from the ossicles using the small scissors, using the spatula and Urs, remove the carpus over the first centimeter of the rostrum. Next, using small scissors, make a horizontal cut across the most rostral exposed tissue in the rostrum to detach it from the exoskeleton.
Then use the edge of the scissors and or spatula. To carefully roll the tissue coddly just past the level of the oval plates. Starting dorsally.
Use the small scissors to detach the remaining tissue from the face and exoskeleton. Using your index finger, press down on the rostrum To better expose this area, cut as close to the carpus as possible and stop. When you get near where the mouth attaches in the center From a ventral view, the mandible should be easily visible.
Detach the remaining maxilla bed. Locate the soft lip under the mandibles. This lip must be kept attached to the stomach while it is detached from the face.
Find the slightly curved bluish outer mouth part and use it as a guide to make a lateral cuts detaching the lip from the face by cutting between the soft lip and the outer mouth part. Now the face can be removed. Follow the next steps to excise the stomach.
Begin by holding what remains of the lobster in the palm of your hand. Ventral side up and rostral end toward your elbow. Now attach a hemostat to the lip and let gravity pull the hemostat and lip down and away from the ventral body wall.
Using the small scissors, detach the stomach wall from the mandibles and surrounding tissue. Locate the dorsal side of the ventral body wall, which forms a hard shelf ventral to lip and stomach. Detach the stomach from this body wall by cutting very close to the body wall below the lip cut across the entire medial lateral midline.
The entire ventral side of the stomach should now be detached from the body wall. Now flip the lobster over to work on the dorsal side. Cut along the rostral edge of the cervical groove where the carpus was removed and gently pulled the stomach out.
Some intestines may come along too, and if they do use scissors to detach them from the stomach. All that's left to do now is to open the stomach and pin it down. Hold the stomach in the palm of your hand with the hypodermis side down.
The hemostat should still be attached to the lip and should lie between your fingers. Now use the small scissors to make a shallow incision along the AP axis at the mouth opening, which is ventral to the lip. Extend the incision down through the ventral side of the pylori.
To further open the stomach, make two lateral cuts along the ossicles running across the center of the stomach. To improve visibility of the stomach interior, rinse it out with cold saline. In order to get the preparation to be flat in the dissecting dish, use the small scissors to trim the three gastric teeth inside the stomach.
Place the stomach hypodermis side up in a saline filled dissection dish. Place insect pins at the lip and the four corners created when the stomach was opened up for the anterior corners. Place pins high and close to the edge as not to disturb the commis ganglia or inferior esophageal nerves for the posterior corners.
Place pins anteriorly to the laterally running ics so as not to disturb the p dilator muscle. Now the preparation is ready for fine dissection of the nervous system. We have just shown you how to perform a gross dissection on the stomach of the American lobster Homa Americana.
This gross dissection of the stomach is the first step in isolating the somatic gastric nervous system. The next step is to do a fine dissection of the nervous system off of the stomach to see how this is done. Please see our accompanying video protocol in JoVE Once isolated.
The somatic gastric nervous system can be used to study various aspects of nervous system function such as the generation of rhythmic behavior. Thanks for watching and good luck with your experiments.
