To make the electrode assembly for multichannel in vivo recordings, four differential electrodes are created and spun together with the individual differential electrodes branching apart. At the ends, the anesthetized animal is pinned out and an incision is made to the side of the eye spot. The target nerves and muscles are located and electrodes are attached to the drop of superglue at the base of the hook, and then insulated with quick seal sealant.
Hi, I'm Miranda Collins from the laboratory of Dr.Halal Thiel from the biology department at Kiss Western Reserve University. Today I'll show you a procedure for assembling and implanting electrodes for in vivo recordings in SIA, California. We use this procedure in our laboratory to study the neuromuscular control of feeding and sia.
Let's get started. To create the electrode use scissors to cut a piece of enamel coated 0.001 inch diameter stainless steel wire about two feet long. Attach a small ball of putty to each end of the wire.
Fold the wire in half and spin the ends to create a twisted pair called a differential electrode. Tape both ends of the wire to prevent unraveling. Make four differential electrodes using a different color of tape for each differential electrode.
For identification purposes, twist two of the differential electrodes together, leaving two inches untwisted on one end and one inch on the other end. Repeat with the remaining pair of differential jodes. Twist both sets of two differential electrodes together, making sure that the ends remain untwisted.
Hold up the electrode assembly and apply household silicone glue at the junction of the four differential electrodes, leaving the ends free. Hang and allow the glue to dry for at least four hours. To insulate the branches, lay the electrode assembly down on a flat movable surface such as a piece of wood.
Use a small piece of plastic to elevate the electrode assembly just before it branches, and secure that with tape. Fan out the ends and secure them with tape. At the dissecting microscope, use forceps to coat the branches with silicone glue.
The one inch long ends will be inside the animal, so they should have a very thin coat of glue. Allow the glue to dry for at least three hours. Shorten the one inch long end of one differential electrode to two centimeters on each end of the electrode.
Use forceps to scrape silicone and enamel away from both wires. To reveal one centimeter of bare wire curl one bare wire into a hook for recording. Then the other wire up and away from the hook to serve as the ground wire on the two inch long end solder a gold connector pin to each wire.
Use a piece of tape to secure the end and keep space between the ground and recording pins. Repeat these steps so that there are hooks and gold connectors on all four differential electrodes. Attach a one to two centimeter ball of the silicone glue to the cable about 1.5 inches from the hook end.
To anchor the electrodes within the animal allowed to dry for at least one hour, select a healthy animal that weighs about 350 to 450 grams in response to small pieces of seaweed. The animal should generate bites at intervals of between three to five seconds. The complete surgery with electrode implantation should take about an hour.
Lay the anesthetized animal out in a dissection tray with its foot down facing the animal head on. Shift the gill area to your left such that the animal is partially on its side leaning away, pinned down the anterior tentacles. Place the tray on the stage of a dissecting microscope.
Prop up the tray at about a 20 degree angle to elevate the head and collect the hemolymph in the tail away from the incision using dissection scissors, start an incision slightly to your right of the eye spot and extend it one to 1.5 centimeters towards the head. Use your tractors. Attach to the edges of the tray to hold the incision open.
To expose the buccal mass. Use forceps to lift the clear membrane underneath the skin and use scissors to cut an opening, matching the main incision. Tuck the edges of the membrane into the retractors.
Place the electrode assembly on a micro manipulator and move it over the animal's incision. Each of the four electrodes will take about 10 to 15 minutes to attach access to the nerves will be shown first followed by attachment to the electrode to the final nerve. However, in practice, each nerve should be attached to an electrode before the next nerve is exposed.
The ular nerve is most difficult to access and should be approached first. There are two options for accessing the ular nerve. The first option, which is preferable, is to look under the buccal ganglion between BN one and VN two.
If a branch of the ular nerve is visible, use fine tipped forceps bent into a hook to lift out the branch. If a branch of the ular nerve is not accessible above the muscle, a second option is to access it via a small incision in I two next to the connective tissue at the base of the buccal ganglion. The incision in I two must go through two layers of the I two muscle to attach an electrode to I two.
Use the hooked forceps to lift and separate two bands of the I two muscle access buccal nerve two here. Buccal nerve three is here and will be used to demonstrate electrode attachment. All the electrodes are attached in the same way as demonstrated now on buccal nerve three.
Once the nerve is hooked on the forceps, use another set of forceps to manipulate one electrode hook onto the nerve using the micro manipulator. Raise the hook so that the nerve lifted up and away from the buccal mass. Clear of any other nerves or tissue lifted as little as possible, half a centimeter or less.
To avoid overstretching the nerve, use a kim wipe to dry off the electrode and adjacent area of the nerve. An insect pin is used to apply the glue, secure the electrode with a small dab of superglue where the base of the hook is in contact with the bottom of the nerve. Apply a drop of alysia saline or hemo lymph to set the super glue.
Then red dry the hook on an ice pack mix. Quick seal glue. This keeps the glue workable for about 30 minutes.
Additional mixings may be necessary over the course of the surgery. Use the insect pin to completely cover any exposed wire of the hook with a small amount of quick seal. Avoiding the ground wire.
Allow the glue to set for five minutes. Readjusting the glue. If it pulls away from the hook, wait until the glue is dry before accessing the next nerve.
Once all the electrodes are attached, slide the electrode assembly into the animal up to the silicone anchor. Suture the incision closed around the protruding electrode cable. Keeping the anchor inside the animal super glue the stitches to ensure a good seal.
Insert the gold connector tips at the other end of the electrode into a small block of styrofoam. Return the animal to the main tank in an aerated isolation container for recovery overnight. Acclimate the animal in an aerated container while attached to the recording equipment for at least 1.5 hours.
Monitor the signal while the animal is acclimating to verify that all electrodes are working and that noise levels are low. Use a video camera to record feeding behavior while using the computer to record signals from the neural and muscular electrodes. Synchronize the video and electrode recordings using a square wave signal that controls a digital counter in the camera view while it is simultaneously recorded as an additional channel on the computer.
In aScope when the animal is feeding, there will be bursts of activity on the nerves and muscle. Here's an example of a swallowing pattern with EMG activity from I two and ENG from RN BN two and BN three. We've just shown you how to fabricate and implant multichannel electrodes to record the neural and muscular activity during feeding behavior in freely behaving sia.
When doing this procedure, make sure to handle the nerve carefully and make sure to completely seal the recording hooks such that none of the wire is exposed. Thanks for watching and good luck with your experiments.
