The overall goal of the use of cooling-induced deactivations in this study is to assess cortical contributions to obstacle memory in the walking cat. This method can help answer key questions in the field of locomotor control, such as whether the parietal cortex is involved in memory-guided obstacle avoidance. The main advantage of this technique is the ability to reverse the deactivation.
This allows each animal to serve as its own control. To begin, construct the apparatus shown here as described in the accompanying text protocol. Next, place the animal onto the testing apparatus while wearing the testing harness, to acclimate the animal to the setup.
This leash orientation allows the animal to walk along the central portion of the apparatus without any tension, thus encouraging the animal to remain within this portion of the apparatus. Establishing such boundaries is helpful when working with a moving test subject. Place soft food onto the food platform and then set the animal onto the walkway, allowing it to eat from the platform.
Ensure that the animal is comfortable with handling, including instances where the animal must be moved to the start area of the walkway. In order to assess visual obstacle memory, raise the obstacle onto the walkway and place the platform on the far side of the obstacle. Then set the animal at the start area of the walkway and allow the animal to approach the food, stepping over the obstacle with only its forelegs in order to eat from the platform.
As the animal continues to eat, lower the obstacle such that it becomes flush with the walkway to prevent any further visual or tactile inputs. Following a variable delay period, move the food forwards again to encourage the animal to resume walking. Also perform trials where the obstacle is absent in order to prevent habituation to the obstacle and development of a learned avoidance response.
Observe hind-leg stepping in obstacle-present and obstacle-absent trials to verify typical locomotor behaviors and intact visual obstacle memory prior to cooling. Ensure that the animal can clear the obstacle without contact and that stepping of all four legs is significantly elevated in obstacle-present trials in comparison to obstacle-absent trials. To assess tactile obstacle memory, begin by ensuring that the obstacle is not raised onto the walkway.
Then place the animal at the start of the walkway and allow it to walk towards the food platform placed on the far side of the obstacle slot. As the animal eats, raise the obstacle onto the walkway beneath the food dish, preventing any visual input of the obstacle. Then move the food forward.
As the food is moved forwards, the animal should contact the obstacle with their forelegs before stepping over it. Allow the animal to continue eating while straddling the obstacle between their fore and hind legs. During this time, lower the obstacle so that it becomes flush with the walkway to prevent any further visual or tactile inputs.
Following a variable delay period, move the food forwards once again to encourage the animal to resume walking. In tactile obstacle-absent trials, have the animal approach and eat from the food platform. However, raise and lower the obstacle before moving the food forward with a variable delay.
Perform trials where the obstacle is absent and no foreleg contact occurs, to prevent habituation to the presence of the obstacle or the development of a learned avoidance response. Observe the hind-leg stepping in the obstacle-present and obstacle-absent trials to verify normal locomotor behaviors and intact obstacle memory prior to beginning cortical cooling. To deactivate discrete regions of the brain and to assess their contributions to memory-guided obstacle avoidance, implant CryoLoops bilaterally over areas five and seven according to previously reported surgical procedures as described in the accompanying text protocol.
Prior to testing, prepare the cooling circuit by combining 200 milliliters of methanol with 500 cc of dry ice to form an ice bath. Connect the intake tube from the methanol reservoir to the pump. Connect the outflow tube that runs to the ice bath to the other side of the pump.
Connect the tubing ends to a dummy CryoLoop and ensure that the ends fit snugly over the inlet and outlet tubes to complete the cooling circuit. Next attach the thermocouple plug to a digital thermometer for continuous temperature monitoring. Ensure that the length of this cable and the tubing are sufficient to reach the head of the animal when connected.
Then turn on the piston pump using the switch and verify that all connections are snug and no leaks are present. Ensure that the pump setting, length of tubing inside the ice bath, and length of tubing from the ice bath to the dummy loops are optimal such that the dummy CryoLoop temperature can reach a steady state below 5 degrees Celsius. Once satisfied with the initial setup, switch the pump off and remove the dummy CryoLoop.
Place the animal on the testing apparatus and buckle the harness. Then secure the strap and attach the leash. Next, remove the protective cap of the implanted CryoLoop to expose the inlet and outlet tubes.
Fit tubing ends snugly over the inlet and outlet tubes of the CryoLoop, and connect the thermocouple plug to the digital thermometer. Begin the testing session with a visual or tactile obstacle memory trial. After the initial test, follow with additional trials of all four types in a random fashion.
Next, switch on the piston pump and wait one to two minutes for the CryoLoop to reach a temperature of three degrees Celsius. Then run a cool block of trials. During the trials, ensure that the temperature of the CryoLoop is maintained around three degrees Celsius.
Then switch off the piston pump and wait for the CryoLoop to return to its original temperature. Run a final rewarm block of trials after the piston pump has been switched off. When the behavioral testing is concluded, remove the tubing from inlet and outlet tubes and ensure that the protective caps are replaced.
Be conscious of residual methanol that may drip from the tubing ends and may irritate the animal. Remove the leash and harness and then return the animal to the colony. Finally, use a tube cutter to trim the tube ends to prevent leaky connections on the next testing day.
When area five of the cat's brain is bilaterally cooled, hind-leg stepping was significantly attenuated in the obstacle-present trials for both the leading and trailing hind legs. The peak step height of the forelegs in the obstacle-present trials and all of the legs in the obstacle-absent trials were not affected by area five deactivation. Similarly, the peak step height for any leg in either obstacle-present or obstacle-absent trials did not differ from the warm condition when area seven was deactivated.
In comparison to both warm and area seven cooled conditions, step clearance was reduced in the leading hind-leg step and in the trailing hind-leg step for the area five cooled condition. Additionally, the step trajectory of the trailing hind leg was affected by area five deactivation as the peak occurred before the obstacle, unlike the stepping in both warm and area seven cooled conditions. While attempting this procedure, it's important to remember to monitor the temperature of cooling loops throughout the experiment to ensure proper deactivation.
After watching this video you should have a good understanding of how to reversibly deactivate regions of cortex with cooling in a behaving animal.
