This protocol is significant, as it provides a direct and cost-effective method for measuring skeletal muscle temperature. Moreover, it can be used in various contexts and under the stimulation of pharmacological agents. The main advantage of this technique is the number of animals that can be tested simultaneously.
The minimal animal disturbance described here, avoids the need for high-tech methods like infrared thermography. Individuals may have difficulty measuring the temperature of the animals within the home cage or on treadmills while in motion. This can be mitigated by practicing measuring techniques during habituation trials.
Begin by warming the enclosed transponders between gloved hands and make sure to maintain sterility, while working with transponders. Measure the temperature using a temperature scanner and check whether the transponder reads the temperature changes. After anesthetizing the mouse as described in the manuscript, use surgical scissors to make a shallow cut through the skin on the right hind limb.
Place the sharp edge of a pre-programmed and uncapped sterile transponder into the incision, parallel to the gastrocnemius. Make sure the green plunger faces up and is visible. Keep pushing the transponder applicator into the incision until the opening of the transponder applicator is no longer visible.
Turn the applicator at 180 degrees, so that the green plunger faces down toward the mouse's limb and is no longer visible to the experimenter. Once the transponder applicator is placed adjacent to or partially enclosed in the gastrocnemius, push the green plunger, allowing the applicator's pressure to guide the investigator's hand away from the mouse. Using forceps, hold the open skin together.
Place a wound clip with a sterile autoclip, bend if required. Use absorbable sutures before the sterile autoclip to close the fascia layer. Using the transponder reader, check the temperature of the mouse muscle.
After removing the mouse from the anesthesia, place it in a clean home cage on top of a heating pad and ensure that the home cage includes a tea ball with an odorless towel to begin habituation. Assign a location to the riser in the testing room, and to avoid confounding variables, separate the risers set to receive different contextual stimuli by a minimum of two meters. Using magnetic strips, attach surgical sheets across the riser and create a visual barrier to minimize temperature fluctuations due to mouse activity, in response to the experimenter's movement.
Prepare tea balls with control and predator odor towels. Transfer the animal to the prepared testing room. Place the animal in a preassigned location on the riser and avoid changing the riser's location between habituation and testing procedures.
Remove the home cage ball from the mouse's home cage. Recover the cage with a cloth or surgical sheet and allow the mouse to acclimate to the testing space for one to two hours. After acclimation is complete, use the scanner to measure and record the baseline temperature of the subject.
Uncover the cage and place the tea ball onto the floor of the home cage. Replace the cage lid and cloth covering. Start the stopwatch and measure the temperatures of the test subjects in the same order of tea ball placement.
Record the temperatures and clock time of measurements, following the desired time points. Prepare the treadmills for testing, ensuring the shockers are functional. Ensure that the animals use the same treadmill for habituation and testing procedures.
Move the mouse to the testing room, and allow it to acclimate for one to two hours in its home cage. Before moving the mouse to the treadmill, measure and record its baseline temperature. For easy placement and removal, adhere the control or predator odor towels to the ceiling or underneath the front of the treadmill.
Guide the mouse into the assigned treadmill. Turn on the treadmill belt and shocker, start the stopwatch, and take measurements of the test subjects in the same order they were set up in the treadmills. Record the temperatures and clock time of the measurements, following the desired time points.
Once the test is complete, turn off the shocker and treadmill and return the mouse to its home cage. Clean the treadmill using liquid detergent and water and remove any residual predator odor. Repeated habituation trials significantly decreased the muscle temperature of mice, showing mice habituated to the testing environment and protocol.
The combined sex analysis of trial four showed no significant difference between before move and baseline temperature measurements, demonstrating the effectiveness of one hour acclimation to the testing context. Pharmacological stimulation using oxytocin in mice showed decreased muscle temperature relative to baseline with a maximal decrease after 30 minutes. Muscle temperatures were normalized 60 minutes after oxytocin injection.
A sustained increase in muscle temperature was observed in Sprague Dawley rats, after removing predator odor as contextual stimuli. Sprague Dawley rats displayed a robust increase in temperature in response to ferret odor, compared to control odor. In the presence of other aversive odors, ferret odor produces and maintains a robust thermogenic change compared to all other conditions.
While attempting this procedure, it is critical to habituate the animals to the testing protocol and to schedule one to two hours of acclimation each day of testing.
