This procedure begins with a gross dissection of the crab stomach in which the appendages and dorsal carpus are removed from the crab. The entire stomach is dissected from the animal. The stomach is cut open and pinned flat to a black sard dish.
In preparation for the fine dissection, the Steamatic gastric nervous system is dissected from the stomach and is transferred to a clear Sard coated Petri dish where it is des sheathed in preparation for an electrophysiology experiment. Hi, my name is Rachel Au from the Martyr Lab at Brandeis University. And I'm Gabrielle Gutierrez, also from the Martyr Lab.
Today we will show you a procedure for dissecting the somatic gastric nervous system from the Crab Cancer Borealis. We use the crab St NS in our laboratory for our electrophysiology, immunohistochemistry, and cell culture experiments. Using this neural network, we can investigate several fundamental principles of neurobiology, such as neuromodulation homeostasis, network variability, network development, and network recovery.
So let's get started. Before starting the dissection, the crab must be numbed by burying it nice for 30 minutes. Be sure to surround it on all sides with ice.
The ice steals the crab's, sensory and motor systems, but the crab might still be responsive when removed, while the crab chills. Prepare for the gross dissection by assembling the equipment required for this part of the protocol. The dissection pan Ron jaws bone cutting scissors, small scissors tooth forceps, a spatula with a tapered edge, a black sill guard coated dish, and insect pins Before beginning.
Prepare yourself by dawning gloves. Once 30 minutes have elapsed, remove the crab from the ice bucket by its hin leg. To avoid being pinched and place in the dissection pan first, remove the claws by twisting each one inward.
Next, remove each leg using the same twisting motion. Now that the animal is appendage list, use the Ron Jos to remove the maxilla covering the mouth and the soft maxil Using the Ron Jos again, start from the lateral posterior end of the carus and get a good grip on the edge of it. Clamp down with the Ron Jos and break that piece of carpus off.
Make sure that you remove enough of the carpus to allow the spatula to enter the opening. Continue removing the edge of the carpus up to the eyes and repeat on the other side. With the tapered side of the spatula, gently scrape the soft inner tissue away from the roof of the dorsal carpus.
While you do this, keep the spatula in contact with the carus. To avoid damaging the STNS, use the Ron Jos to break away parts of the carus that have been separated from the tissue. Break away the dorsal carus up to where there are two small S protruding from the carus.
Cut as far anteriorly as possible. Stabilize cephalon with one finger while breaking away the most anterior parts of the dorsal carus. Separate some of the soft tissue from the ventral carus.
Make sure to separate away any adductor muscles attaching the tissue to the carus on the ventral side of the carus. Pull out the mandibles one at a time by anchoring the wrong jaws onto a mandible twisting outwards and pulling away two long obstacles with muscles should be attached to the extracted mandible. If done properly, separate away the tissue from the sides of the cephalon near the eyes.
Using the spatula with small dissection scissors, layer by layer cut across the tissue right next to the ridge of ossicle. Prop up what remains of the crab against the dissection pan. Using the detached claws, detach all of the labrum from the epistle.
Using the small scissors using bone cutting scissors, make two diagonal cuts into the ventral carpus. To remove the cephalon, now take the labrum with tooth forceps and cut the labium away from the osci on the ventral side. Gently lift the lip away from the remaining ventral carus.
Use small scissors to separate the ventral part of the stomach from the tissue lining the carus. When the pori canula are visible, cut the stomach away from the tissue on either side of it until the stomach is extracted from the crab, holding the stomach by the lip with forceps. Rest the stomach against your palm and pour saline into the mouth.
This will expand the stomach and make it easier to cut open using the small scissors. Cut from the opening of the mouth down between the ula right through the obstacle. Then cut diagonally down through the two cardio branches.
Lastly, cut off the tips of the three teeth inside the stomach. Place the stomach onto a black sard coated dish with the inside of the stomach down to prepare for the fine dissection. Pinned down the five corners of the stomach with insect pins and immerse the preparation in cold saline.
The fine dissection is done under the stereoscope and requires forceps and spring scissors, as well as a pin holder with a tungsten needle. A clear sill guard coated Petri dish and some fine wire pins should already be prepared beginning on the lowest magnification, pin down the flaps near the lip that used to be the mouth opening. Now make the preparation taut by rearranging and adding pins as needed.
With forceps, carefully lift the spotted hypodermis covering the lower part of the preparation and trim it across What should remain as a patch of hypodermis with two white dots. Now peel up the yellow wispy membranous tissue near the bottom of the preparation. The M vns are weak attached to the underside of this tissue.
Using the blade of the scissors carefully separate the M VNS from the yellow tissue as it is pulled up. Cut away the yellow tissue alongside the few visible thick white processes of the brain. Most of the brain is currently buried in tissue.
Follow the processes that extend laterally and toward the lip. These are the two commissural nerves. Once the end of the commissural nerve is found, remove the massive spongy off white tissue that has been cut away from the side of the brain.
Locate the iOS and sos at the junction where they meet the commis ganglia. Cut away the muscle and tissue to reveal the length of the IO ns and so ns, these two nerves run medially from the commis ganglia and meet the esophageal nerve. 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 underneath them to separate them from the artery. This step exposes the ALNs.
Remove the flanking muscles as far up as the cartilage line over the STN. The TN is a nerve running up from the STG and it has a slight bulge. Next, cut away the arterial tissue surrounding the STG down to where the M vns DGN and DVN meet.
Follow the DVN as it splits into the two LVNs. Cut through the whiteish delicate tissue until each LVN is completely exposed and meets the DLVN. Locate the PSN over the Osco.
Near the bottom of the preparation, the PSN can be used to locate the LVN and DLVN junction. So the DV DLV goes this way. It comes down here.Yep.
Then it splits and goes this way.Right. And so what I, what I normally do is I find them down here and I sort of work my way back up The PDN forks off of the DLVN and is between the ULA and the cardio Pylori valve muscles.Yeah. So here are the ampule ula, ula ampule, the P, the PYN right here.
Be sure to leave some muscle attached to one of these nerves as a marker to identify them from each other. Once all of the nerves have been uncovered, sever any remaining connections between the STNS and the stomach. Finally, carefully move the STNS away from the rest of the stomach.
Cutting any missed connections. Prepare the clear S guard dish by rubbing some stomach tissue over it until it feels wet and less sticky. The STNS will adhere strongly to the sogar.
If the dish is not conditioned in this manner, then add some fresh cold saline to the dish. Grab the upper ends of the commissional nerves with forceps and move the st NS to the clear sogar dish. The brain will still be attached to the commissional nerves to prevent untangling.
Now, prepare the STNS for pinning by severing the brain from the Commis nerves. Use minuchin pins to secure the four ends of the Commis nerves to the sill guard. Ensure that the preparation is right side up by checking that the IVN is pointing up and that the DGN exits slightly beneath the STG pin.
The rest of the nerve ends down with the fine wire pins Using fine forceps and scissors. Carefully clean away any remaining muscle or tissue from the STNS. The STG must now be de sheathed using a pin holder in a slightly hooked fine tungsten needle.
Make a small hole in the corner of the STG sheath away from the cell bodies. Gently poke the tungsten needle into the hole and get it between the sheath and the Samara. Cut or gently tear a flap of separated sheath to expose the STG neuro pill and cell bodies.
Pin any flaps of sheath down to increase accessibility to the cell bodies and to stabilize the STG for intracellular recordings. Rein the rest of the STNS ensuring that each nerve is taught and well spaced for extracellular recording. Ideally, all of the nerves should be free of necks and damage, particularly the ones you will record from.
None of the nerves should be tangled or twisted. The STG should be intact with all of its cells arranged in a beard formation around the NI bill. The TNS was bilaterally symmetrical and looks like a homunculus with the LVNs as the legs.
The M vns is the arms and the anterior end is the head. We've just shown you how to dissect the somatic gastric nervous system of the crab in preparation for an electrophysiology experiment. When doing this procedure, it's important to remember to dissect the nerves you plan to record from by following the desired nerves into the muscles they're known to innervate.
Ensure that the STNS is pinned taut onto the clear cigar dish with the de sheathed side of the STG facing up. So that's it. Thanks for watching and good luck with your experiments.
