Our protocol helps answer the questions of how physical exercise supports organism homeostasis. The systems allowed us to dissect direct mechanical effects on the brain from complex consequences of physical activities such as walking and jogging. To begin, fix the accelerometer on top of the head of a rat using surgical tape.
After complete recovery from anesthesia, place the rat in the treadmill machine. Adjust the treadmill to a moderate velocity at 20 meters per minute. Use the application software to measure the magnitude of vertical accelerations along X, Y, and Z axis when the rat is running the treadmill.
Match the values obtained from the software to the magnitude in frequency of vertical acceleration in the PHM system. Preset the amplitude of oscillation of the platform and the rotation speed of the propeller shaped cam. Then place the mouse in a prone position with the head located on the oscillatable and the rest of the body on the static platforms.
Turn on the motor to oscillate the platform vertically and apply PHM to the mouse. In the transparent plastic case, set up the video camera at a frame rate of 24 FPS to record the entire space. Next, intraperitoneally administer five hydroxytryptophan to a mouse.
Place the mouse in the transparent cage and start recording for 30 minutes. Review the recorded video at 0.5x speed and count the head twitching manually. Retrieve the cryosections of the mouse brain from the slide box.
Leave the slides on clean wipes at room temperature until the samples dehydrate entirely. Use a liquid blocker pen to draw a circle around the cryosectioned tissue. Then add TBS-T solution.
On the bottom of a tray, place dampened wipes to create a moist environment for the slides. After a permeablization with TBS-T, add 4%donkey serum for blocking and incubate at room temperature for one hour. Rinse the slides once by immersion in TBS-T for five minutes.
Then apply 100 microliters of appropriately diluted primary antibody and dappy mix on each slide. Cover the tray and incubate at room temperature overnight. After incubation, rinse with TBS-T thrice for five minutes each.
Apply 100 microliters of appropriately diluted species matched fluorescent secondary antibody and incubate at room temperature for one hour. Afterrinse with TBS-T thrice for five minutes each, mount the slides with mounting medium and cover the slides with cover slips. After mounting, view the sample under a fluorescence microscope.
The PHM system was adjusted to produce vertical acceleration peaks at approximately 1.0 times G which was equivalent to those of the heads of rats during treadmill running. PHM application to mice significantly attenuated their head twitch response as compared to the control mice representing a suppressive effect of PHM on 5-HT2A receptor signaling in the prefrontal cortex neurons. Treadmill running in PHM significantly enhanced 5-HT2A receptor internalization and mouse prefrontal cortex neurons.
Quantification of internalized and membrane associated 5-HT2A receptor positive areas relative to the new and positive area confirmed this observation. Immunosstaining and quantification of c-Fos expression in 5-HT2A receptor positive neurons showed that treadmill running in PHM downregulated 5-HTP induced c-Fos expression. This is the downstream cellular event of 5-HT2A receptor activation and mouse PFC neurons.
Mechanical intervention can be applied to the body parts other than the head to investigate the mechanical aspects of exercise effects.
