The overall goal of this procedure is to record electrical activity from the nerves, muscles, and neurons, and the corresponding movements of the feeding apparatus during feeding motor programs. This is accomplished by first removing the buccal mass from an anesthetized plegia and attaching hook electrodes to key nerves and muscles. The second step is to place the buccal mass in a recording dish and prepare the ganglia for extracellular recording.
Next, the target neurons are located using extracellular electrodes. The final step is to induce and record feeding motor programs such as biting, swallowing, and rejection. Ultimately, the suspended buccal mass preparation is used to show the activity of nerves, muscles, and individual neurons during different feeding like movements of the buccal mass.
The main advantage of this technique over existing methods like isolated ganglion studies, is that recordings from nerves, muscles, and individual neurons can be directly related to the movements of the feeding apparatus. Helping demonstrate the procedure will be huu, a grad student from our laboratory To begin the procedure anesthetize a 200 to 300 gram healthy plegia in a dissecting tray by injecting about 50%body weight of isotonic magnesium chloride. After the first injection near the tail of the animal, gently massage the surface of the animal to spread out the magnesium chloride.
Give additional injections near the head of the animal as needed. After the animal stops responding to gentle tactile stimuli, pin it to the tray with the dorsal side up. Place one pin in the tail and one pin in each anterior tentacle.
Then use the forceps to pinch and lift the animal's skin in the middle of the head. Behind the rhino fours make a coronal incision across the animal's head just behind the point being held. After that, make a mid sagittal incision in between the rhino fours towards the mouth to expose the buccal mass.
Next, pull the flap of skin away from the buccal mass and cut through the nerves and connective tissue that attach to the body wall. In order to fully separate this flap of skin from the buccal mass, be sure not to cut any nerves or tissue intrinsic to the buccal mass. Afterward, grab and hold the esophagus.
Cut through the esophagus posterior to the point being held. Pull up the esophagus and lift the buccal mass behind the buccal mass. The cerebral, plural, and pedal ganglia form a ring with the cerebral ganglion attached to the buccal ganglia by the cerebral buccal connectives.
Leave these ring ganglia attached to the buccal mass and cut all the nerves projecting from these ganglia to other parts of the body. Then cut through the connective tissue around the buccal mass and continue to lift the buccal mass up as it becomes less connected to the body. Once the buccal mass is attached only to the mouth, hold the esophagus and the buccal mass.
Straight up. Make a final cut just anterior to the jaws to free the buccal mass from the body. Place the buccal mass in a solution of 50%isotonic magnesium chloride solution, and 50%SIA saline in order to maintain partial anesthetization while electrodes are attached.
Subsequently, place the buccal mass with the solution in a Petri dish with cigar bottom cut through the buccal artery on the ventral surface of the buccal mass. Then remove the pleural and pedal ganglia by cutting their connections to the cerebral ganglion. Leave the cerebral ganglion attached to the buccal ganglia and the buccal mass via the CBCs and sever other connections between the cerebral ganglion and buccal mass.
Trim the esophagus and salivary glands to short lengths. In this step, attach the hook electrodes to the ular nerve eye, two muscle buccal nerves two and three, and branch A of buccal nerve two. Using a procedure similar to that demonstrated in the JoVE publication by Collins and Shield, and briefly summarized here.
For each electrode detachment, position the electrode near the nerve or muscle, placed the target nerve into the hook and gently lift using a manipulator. Then dry the section of nerve using a Kim wipe. Apply a glob of quick gel super glue to the hooked nerve, and make sure the hook is completely covered with glue and the tip of the reference wire is not covered with glue.
Next uses syringe with a polyethylene tubing attachment to apply solution from the dish onto the glue, which will induce the surface of the glue to set. The next step is to prepare a round 100 by 50 millimeter Pyrex recording dish to use As a recording chamber, apply a thin layer of vacuum grease to the notch between the cerebral and buckled chambers with a pipette tip in order to minimize leakage. Then fill the front chamber with a plescia saline.
Carefully transfer the buccal mass from the Petri dish to the front chamber of the round Pyrex dish, and to make sure that none of the electrodes are pulled tightly, which could break the nerves. Be very careful with the hook electrodes if the dish must be transferred to another binocular dissecting microscope. For thinning the sheath.
Group the electrodes on each side of the buccal mass together. Carefully hold the electrodes by grasping the lab tape that covers the connector pins, and again, make sure that none of the electrodes are pulled tightly. When the dish is positioned under the microscope, the electrodes should be draped gently over the sides of the dish and rest on the platform during breaks and between stages of the experiment, aerate the saline in the buccal mass chamber using an aquarium airstone.
Next pin the cerebral ganglion in the back chamber with the CBCs running through the notch. Apply more vacuum grease over the CBCs. Then add more SIA saline to both parts of the dish so the ganglia are completely submerged.
Be sure that the saline level is lower than the height of the vacuum grease between the cerebral and buccal chambers so no leakage occurs between the chambers. At this stage, the buccal mass should rest at the bottom of the front chamber. Next pin the buccal ganglia on the middle platform to avoid damaging the nerves that are still attached to the buccal mass.
Only place pins on the sheath between two nerves. Then add two more pins on the side of the CBCs to stretch and anchor them. Keep the buccal ganglia flat or rotated based on the location of the neurons of interest.
To rotate the buccal ganglion, use a pair of fine forceps to grab some excess sheath of the CBC and pin it down between buccal nerve two and buccal nerve three. Then most of the cell bodies that are proximate to the back chamber and cannot be easily accessed from the top of the buccal ganglion can now be seen. Lastly, add two pins on the sheath of the buccal ganglion proximate to the buccal mass side.
To minimize the movement of the buccal ganglion, grab the sheath of the buccal ganglion proximate to the back chamber and cut away the excess sheath with fine scissors without exposing the cell bodies. In order to minimize damage, only remove the amount of sheath necessary to see the cell bodies after thinning of the sheath is completed. Attach all the electrode pins to their sockets on the cables that connect to the amplifiers.
Make sure that the electrodes are not pulled tightly and are correctly attached to their appropriate cables and that the polarities are correct to wash out any remaining magnesium chloride. Replace the SIA saline in the buccal mass chamber with fresh SIA saline. Next, thread a silk suture through the soft tissue at the front of the buccal mass and dorsal to the jaw cartilage.
Use two pieces of modeling clay to attach the suture to the edge of the dish, thus suspending the buccal mass from its anterior end position. A manipulator holding a glass extracellular electrode so that the electrode tip is near the buccal ganglion. To locate and identify a neuron, gently lower the tip of the extracellular electrode onto the sheath.
Over the neuron soma. Apply a stimulating current and then switch the channel that is being used to excite the soma to recording mode in order to record the activity on the extracellular electrode and corresponding activity on the nerves to induce rejection. Like motor programs stimulate buccal nerve two A with a long train of two hertz, one millisecond pulses to generate ees patterns.
Patterns continue for the duration of stimulation and may continue until shortly after the stimulation ends. To induce biting like motor programs. Place a few crystals of solid caracol directly on the cerebral ganglion.
Repetitive patterns generally begin within five minutes and last for roughly 10 to 15 minutes before beginning to run down to induce swallowing. Like motor programs, apply caracol and wait until the buccal mass generates strong bites. Then during a bite, place a strip of seaweed in the animal's mouth so that the grasps the seaweed.
Here is the stimulation and recording of the identified motor neuron B nine and the recording from the same neuron, the I two muscle, and several nerves during feeding motor programs. Here are the video images taken from the same series of motor programs. Swallowing patterns were induced by application of carbahol to the cerebral ganglion and the subsequent placement of a seaweed strip into the grasper during carbahol induced biting.
In this image, the ragula is protracted to grasp the seaweed, and in this image, the ragula has retracted pulling seaweed into the mouth. After watching this video, you should have a good understanding of how to set up the suspended buccal mass preparation, and obtain recordings of the in vitro feeding motor programs.
