Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

The overall goal of this procedure is to extracellularly identify individual motor neurons for a motor pool, then to monitor and control them during motor patterns. This is accomplished by first placing the prepared buccal mass of plegia with hook electrodes attached to key nerves in a specialized recording dish, and then preparing the buccal ganglia and cerebral ganglion for extracellular stimulation and recordings. The second step is to attach suction electrodes to the key nerves and muscles of the buccal mass to attach forced transducers to the target muscle and to place extracellular glass electrodes onto the sheath of the buccal ganglia.

Next, locate the target motor neurons with the extracellular glass electrodes. The final step is to extracellularly, stimulate and record target motor neurons while the muscle contraction forces are measured simultaneously. Ultimately, the extracellular stimulation and recording technique is used to show the identification of individual motor neurons and their neural activity during different feeding like motor patterns.

The main advantage of this technique over existing methods like intracellular identification of neurons, is that extracellular glass lectures can identify and monitor multiple motor neurons in the same preparation during muscle movements. Helping to demonstrate the procedure will be Jeff McManus, another graduate student from my laboratory In this procedure to prepare the suction electrodes first cut a piece of polyethylene tubing about 10 centimeters long. Hold the tubing on both ends and place it very close to the flame generated by the bunsen burner while rotating it until it becomes soft.

Then stretch the tubing carefully while moving it away from the flame. The middle part of the tubing will become elongated and narrowed as it is pulled after that, cut the tubing in half to form two section electrodes. These suction electrodes are generally applied to the cut ends of nerves or muscles, though they can sometimes be applied on passant.

After attaching the hook electrodes under the microscope, cut the buccal nerve one and the esophageal nerve bilaterally at the attachment point to the buccal mass. Next, pull the cerebral ganglion forward in order to move it out of the way of the I two muscle. Then cut into the I two muscle over the ular sac.

Extend the cut laterally and anteriorly and pull the flap of the I two muscle forward to expose the ular nerve. Cut the two RN branches and make sure that the branches are long enough for suction electrode attachment. Continue the I two cut in a wide circle around the buccal ganglia.

Being careful not to cut the BN twos or the BN threes until the buccal ganglia and the attached part of the I two muscle can be fully separated from the buccal mass. After that, cut the bilateral BN threes at the attachment point to the buccal mass beyond the hook electrode attachment. Then apply a thin layer of vacuum grease to the notch in the recording dish that connects the back chamber and the middle platform.

Subsequently apply a thin layer of quick gel super glue to the glass bottom of the front chamber where the buccal mass will be placed. Carefully transfer the cerebral ganglion, buccal ganglia, and buccal mass to the recording dish. Make sure that none of the hook electrodes are pulled tightly, which could damage the nerves.

Next carefully, place the buccal mass on the glue in the front chamber of the recording dish. Ensure that its ventral surface is glued to the bottom of the dish and keep the ganglia and electrodes from touching the glue. Afterward, add plegia saline to the dish to induce the glue setting.

Then under the microscope, grab the sheath of the cerebral ganglion and pull it into the back chamber with a pair of forceps and ensure that the CBCs run through the notch. Then pin the nerves of the cerebral ganglion other than the CBCs. To avoid damage to the intact CBCs.

Apply more vacuum grease over the CBCs so that the top of the vacuum grease is slightly higher than the so guard wall and no leakage will occur between the chambers. Then add more plegia saline to both chambers of the dish so that the ganglia are completely submerged. To stabilize the buccal ganglia first pin, the ends of the BN threes on the cigar base of the middle platform.

Since the BN threes will be recorded using hook electrodes, the pins should be placed more distally than the attachment points of the hook electrodes then pin the ends of the BN ones and the ends. Then use two pins, which have been bent 90 degrees as hooks to stretch and anchor the CBCs so that the CBCs will not be damaged. Next, pin down the RN branches between the back chamber and the buccal ganglia.

The I two muscle will then be on top of the buccal ganglia to expose the I two nerve use forceps to grab the I two muscle and pull it over the buccal ganglia and pin two corners of the I two muscle to avoid damaging the I two nerve. Afterward, sever the I two nerve distal to the point where its two branches, innervate the I two muscle and cut away the rest of the I two muscle and flip the I two nerve back pin the I two nerve down between the two RN branches. Next, adjust the locations of the pins in order to stretch and add tension if a nerve is too loose or to release tension if a nerve is too tight.

To further stabilize the buccal ganglia, add more pins on the sheath between the nerves. Since the buccal ganglia are placed coddle side up rotate the buccal ganglia. If the neurons of interest are on the rostral side.

To rotate one of the two buccal ganglia, use fine forceps to grab some excess sheath of the CBC that is near the buccal ganglia. Then pin the excess sheath of the CBC down between the CBC and the BM three, and add an additional pin on the sheath of the buccal ganglion on the side close to the front chamber to minimize the movement of the buccal ganglion. To trim the sheath covering the buccal ganglia.

Use fine forceps to grab the sheath on the side close to the back chamber. Then cut away the excess sheath with fine scissors without exposing the cell bodies. In order to minimize damage, only remove the minimum amount of sheath necessary to see the cell bodies.

After the sheath of the buccal ganglia is trimmed, pull the iTune nerve and the RNs over the buccal ganglia and pin them down between the buccal ganglia and front chamber. To further rotate the buccal ganglia to fill the electrode with aple saline, attach the free end of a 15 to 20 centimeter long polyethylene tubing on the syringe to the end of the glass electrode. Next, pull the plunger of the syringe back to fill up the electrode with a plegia saline.

Place the filled extracellular glass electrode in the notch of the holder on the manipulator. Then use the manipulator to place the electrode tip into the plegia saline containing the buccal ganglia. After that, insert a silver, silver chloride wire into the electrode to service the recording wire and connect it to A BNC cable that connects to the amplifier.

Then attach another wire to the BNC cable to serve as the reference wire with the other end of the wire placed into the plegia saline. In this step, trim the narrow end of the suction electrode tip to match the diameter of the nerve. The inner diameter of the electrode tip should be similar to or slightly smaller than the nerve's diameter to ensure tight suction.

Since the iTune nerve and the RN are very close to each other, place their electrodes in the same manipulator to save space. Rotate the two electrodes and ensure that their tips are close to each other. Then choose one of them for the iTune nerve recording the other one for the RN recording.

Place the electrode tip in the plegia saline. Attach the free end of the polyethylene tubing on the syringe to the suction electrode. Then use the syringe to fill up the electrode with aple saline.

Subsequently move the electrode tip close to the end of the I two nerve and use the syringe to suck the nerve into the electrode. The length of the nerve within the electrode should be about 0.5 to one millimeter. To ensure a tight seal, repeat the suction for the electrode that will be attached to the rn to attach the force transducers to the muscle.

Bend the curved needle of each suture and tie the suture to the force transducer. Gently grab and lift a small amount of muscle with forceps and insert the needle through the posterior part of the I one I three muscle up to the bent point of the needle. Now attach another force transducer to the anterior part of the muscle at the jaws.

Next, lift the force transducers until the sutures are pulled taut, but do not overstretch to check this. View the measurement from the force transducer when the suture has some slack in it. After that, lift the transducer until the measurement is slightly above this baseline level.

To locate a candidate neuron, use the manipulator to gently press the tip of the extracellular glass electrode down onto the sheath over the center of the neuron soma, which is the best location for stimulation and recording cell selectivity. This figure shows the extracellular stimulation of B three and the recording from B three soma, as well as from the corresponding nerves and muscle regions from top to bottom. The channels are the recordings from the B three soma, the contralateral BN two, the ipsilateral BN two, the ipsilateral BN three, the contraction force of the anterior region of the I one I three muscle, and the contraction force of the posterior region of the I one I three muscle.

The blue box highlights the duration of forces in the anterior and posterior regions of the I one I three muscle B three only projects on the ipsilateral BN two. Here are the extracellular recordings from the B three soma and nerves in an EST like motor pattern as well as an ingestive like motor pattern. The blue boxes indicate the protraction and retraction phases of the patterns.

The red bars in the B three SOMA channel highlight the action potentials recorded from the B three soma, whereas the red bars in the IB two channel indicate the corresponding timing when B three is firing in the ipsilateral BN two. During the feeding motor patterns. Note that the BN two unit of B three is the largest of all units.

Thus, we can also detect the BN two units of B three directly from the BN two recordings without SOMA recordings. This figure shows the extracellular stimulation of B 43 and the recording from its soma, as well as from the corresponding nerves and muscle regions from top to bottom. The channels are recordings from the B 43 soma, the contralateral BN two, the ipsilateral BN two, the ipsilateral BN three, the contraction force of the anterior region of the I one I three muscle, and the contraction force of the posterior region of the I one I three muscle.

The blue box highlights the force measurements of the I one I three muscle during B 43. Activity B 43 projects on the ipsilateral BN two only. Here are the extracellular recordings from the B 43 soma and nerves in an ingestive like motor pattern as well as an ingestive like motor pattern.

The blue boxes indicate the protraction and retraction phases of the patterns. The red bars in the B 43 SOMA channel highlight the action potentials recorded from the B 43 soma, whereas the red bars in the IBN two channel indicate the corresponding timing when B 43 is firing in the ipsilateral BN two. In these patterns, note that the BN two units of B 43 are small and very difficult to detect without summer recordings, but fire consistently at the end of the BN two motor program, which provide another way to identify them.

After watching this video, you should have a good understanding of how to extracellularly identify individual modern neurons for multiple.