Long-term Sensory Conflict in Freely Behaving Mice

The overall goal of this protocol is to produce a persistent sensory conflict for the study of long-term learning in freely behaving mice. Lowering the experimental cost and time of sensory learning, this approach accommodates the combination of in vivo and in vitro experiments. After confirming a lack of response to toe pinch in a sedated mouse, use a pair of blunt forceps to grasp the skin at the back of the head and use scissors to make a one and a half cm longitudinal incision to expose the skull.

Gently scratch the periosteum with a scalpel and apply a drop of green activator onto the middle of the skull to increase the bone permeability. Next mix one spoonful of polymer from the implantation kit with five drops of monomer and one drop of catalyzer and brush a generous amount of cement mix between the lambda and bregma skull landmarks. Using a swiping motion from lambda to bregma, quickly place the headpost onto the cement and reapply additional cement around the inferior part of the post to ensure that the headpost sticks securely to the skull.

While the cement is drying, mix the resin powder with resin liquid to a smooth consistency and apply the resulting resin mixture to the dried cement and around the headpost. After allowing the resin to dry for three minutes, use a Vicryl 4-0 suture to close the skin at the back of the ears and use a cotton swab to apply a 10 to 20%iodine solution to the incision. Then place the animal under a red light with monitoring until full recovery.

Forty eight hours after the surgery align the holes in the striped device with the holes in the headpost and use a 1.3 mm hex screwdriver and two 1.2 mm screws to secure the device to the headpost. We recommend fixing the device onto the headpost with the help of a second operator to avoid the risk of ripping off the headpost or hurting the animal with the screwdriver. To fix the sham condition, turn the device upside down and with the back part of the device facing the rostral direction align the holes in the device with the holes in the headpost.

Confirm that the device is well-secured and cannot be removed by the animal and that the device does not apply pressure directly onto the nose, which could potentially cause pain, breathing difficulties, or skin injury. As the mice may exhibit some behavioral abnormalities during the first 48 hours of wearing the device, monitor the animals closely during this time and provide easy access to food and water until the animals become acclimated. Weigh the mice immediately after implantation and again every 24 hours thereafter until the end of the experiment, giving special attention to the mice wearing the striped device.

To record the sensory response of the device-adapted animals, secure the first mouse onto the turntable with the screws inserted into the headpost and place a screen dome around the animal. Turn off all of the lights in the room except for the optokinetic projector and start the optokinetic reflex full field stimulation recording at several different velocities in both the clockwise and counterclockwise directions. As soon as the recordings are over, remove the dome and turn on the lights.

To record the vestibulo-ocular reflex in pitch dark, apply a drop of 2%pilocarpine to the animal's eyes and wait at least five minutes before gently removing the ointment with a cotton swab. Turn off all the lights again and place a box over the turntable to keep the animal in pitch dark. Before starting the horizontal vestibulo-ocular reflex test using sinusoidal angular rotations around a vertical axis with different frequencies and/or different velocities.

When the second recording session is finished, return the mouse to its cage under an infrared lamp with monitoring until the animal is fully recovered from the secondary vasodilator effects of the pilocarpine. After two weeks wearing either a sham or a striped device, the vestibulo-ocular reflex responses of the sham mice remain unaltered after the adaptation, while the striped device-adapted animals demonstrate a drastically compromised reflex response. Quantification of the mean vestibulo-ocular reflex gains at a fixed frequency of 0.5 hertz and at 40 degrees per second before and after the visual-vestibular mismatch protocol reveals a strong gain decrease in the mice that wore the striped device while the sham mice did not exhibit significant gain changes.

However, the optokinetic reflex of mice wearing the striped device is comparable to that observed for the period prior to the visual-vestibular protocol and to that demonstrated by the sham animals. Be sure to monitor the mice daily and to make sure the device is correctly positioned on the mice and that it does not impede their routines. Following this procedure in vitro electrophysiology or neuronal imaging for identifying sellar adaptation mechanisms or behavioral assays for studying different reflex outputs can be performed.