The overall goal of this protocol is to use genetic advances in mice, and our understanding of the whisker system, to investigate the neurobiological basis of eyeblink conditioning, a translatable paradigm for studying learning and memory. This method can help answer mechanistic questions in learning and memory by using calcium imaging and electrophysiology in genetically manipulated mice while stimulating specific whiskers during conditioning. The main advantage of this technique is the ability to secure the heads of mice while minimizing stress, by allowing the mice to ambulate at their own pace on a cylindrical treadmill.
The implications of this technique extend toward potential treatments for memory impairments, including those associated with normal aging and Alzheimer's disease. Preparation of the cylinder and the whisker stimulation system are each covered in the text protocol. This section covers preparation of the headpiece.
The headpiece strip is a 3D printed seven hole strip modeled from the Amphenol 221 series nylon strip that is no longer manufactured commercially. From the 3D printed piece, tap the first hole in the strip for 0-80 by one inch machine screw and leave one hole empty after the first hole. Next, using a vice, put five gold-plated pins in the remaining five holes.
Push the pins through the narrower holes in the plastic. Next, using a thermal stripper, strip a half centimeter of polyimide coating. Then, solder the stripped end to one of the pins where the pin has an opening.
Cut the soldered wire to between six and seven millimeters and strip about two millimeters of the wire. Now, repeat this process to attach four wires in total to the five pins. The first two wires will record eyelid EMG responses while the second two will deliver electrical stimulation to the periorbital region.
To the fifth pin, solder an uninsulated five centimeter stainless steel wire to serve as an electrical ground. After making all the connections, check each of them with a multimeter to ensure their electrical continuity. Begin with a fully anesthetized animal, confirmed by a toe pinch.
Inject a dose of buprenorphine hydrochloride as an analgesic. Then apply a small amount of ophthalmic ointment to the corneas and shave the top of the animal's head. Next, secure the animal's head to a stereotaxic frame and disinfect the scalp with three alternating scrubs of povidone iodine and alcohol.
With a number 10 or 15 scalpel blade, make an incision along the midline of the scalp, exposing the skull from the front of the eyes to past the interparietal bone. Retract the flaps of the skin with six microclips, exposing as much of the skull as possible, including the sides and back. Next, use the scalpel to scrape the periostium off the skull.
Then clean the top of the skull with 3%hydrogen peroxide three times. Once the skull has dried, drill two holes with a size 34 inverted cone bur, being careful not to damage the brain. Place one hole in front of bregma, and place the other hole in front of lambda, left of the midline when conditioning the right eye, or vice versa.
These holes should be drilled carefully to fit the screws, and great effort should be made to not damage the brain. Into the holes, place screws for the electrical connections. Each full turn will lower the screw 0.28 millimeters, so two full turns is sufficient.
Next, create several small divits on the skull in order to increase the surface area for the cement. From the completed headpiece, wind the ground wire in a figure eight configuration around the two screws. Leave some slack in the ground wire so the headpiece can be positioned properly.
Now, apply cold luting cement. Coat the skull and the screws liberally and allow the cement to dry. Once the cement is dried, position the headpiece vertically, with the pins standing up above the skull.
Secure the headpiece with a holder attached to an arm from the frame. The holder must have five gold-plated sockets to accept the pins of the headpiece. Position the two EMG wires on the muscularis orbicularis oculi above the eye socket.
Then, position the two stimulating wires. Put the stripped ends under the skin two to four millimeters directly caudal to the eye. Do not let their ends touch each other.
If the wires are so long that they could scratch the eye of the animal, trim them back or bend them back. Some bare wire must remain exposed. Now, cement the base of the wires to the skull with a little more luting cement and allow it to dry.
Use about half the volume previously used. To proceed, remove the microclips and reposition the skin. Allow the skin to settle naturally to prevent tension on the skin that would distort the eyelid inhibiting blinking and thus ruin the experiment.
Once the skin is settled, remove the headpiece holder and seal the exposed area with dental cement. Avoid dripping cement on the eyes, or the pins of the headpiece. Then use a cotton swab dampened with dental cement solvent to smooth and manipulate partially cured cement as needed.
Once the cement has dried completely, remove the animal from the stereotaxic frame and allow it to recover on a warm surface before returning it to its home cage. Start habituating mice after they have had at least five days of recovery time. To secure the mouse, briefly restrain it by the tail to pinch it behind the shoulders.
Then lift the mouse from under the abdomen and torso. Next, attach a connector to the headpiece and turn the locking screw. Then, place the mouse gently on the cylinder and hold it still while using two screws to fasten the connector to the bars above the cylinder.
Then release the mouse. In the first two sessions, let the mouse habituate to the cylinder for the same length of time as a conditioning session. During the habituation, record the spontaneous blink rate.
Also, pre-expose the mice to the whisker vibration stimulator without applying electrical stimulation. With the piezo system close to the conditioned side, slip the teeth of the comb over individual whiskers. Select the same whiskers for every session.
Then proceed with classical conditioning training as presented in the text. Eight to ten week old male C57 Black 6J mice were trained with trace eyeblink conditioning in the headfixed cylindrical treadmill. EMG recordings of each trial were taken.
The EMGs were rectified and integrated with a 10 millisecond time constant, then averaged across all the trials. The evolution of conditioned repsonses over 10 training sessions shows that gradually the response got larger in amplitude and the response latency became shorter. This evolution is not seen in the responses from the psuedoconditioned mice, where the stimuli are presented in random order.
The evolution of well timed, and thus well learned, conditioned responses can also be seen in the histograms of the time to the peak of the response following CS onset. After watching this video, you should have a good understanding of how to prepare the headpiece, surgically implant the headpiece on a mouse, and place the mouse on the cylindrical treadmill, for headfixed eyeblink conditioning. Your mice should exhibit a good learning curve across days as they acquire the conditioning.
Once mastered, this technique can be done in one hour if it is performed properly. While attempting this procedure, it's important to remember to remain vigilant in observing any distress from the animals. Appropriate steps should be taken in order to minimize stress to the animals.
