These protocols are important for establishing the integrity and normal functioning of the autonomic system to assess autonomic nervous system functionality and to reveal vestibular system disorders. A vestibular functioning or motor coordination deficit can signal damage to the autonomic system. These techniques can reveal the integrity of autonomic functioning.
Behavioral techniques can be susceptible to variabilities introduced by the experimenter. We recommend the same researcher conduct the techniques throughout the experiment and to treat every rodent identically. For elevator vertical motion analysis, place up to four two-month-old 200 to 250 gram rats in a 22.5 by 26 by 20 centimeter plexiglass box and securely fashion the box.
Place the box onto the elevator pad of an elevator vertical motion device and turn on the device at the lowest setting for acclimatization. Next, set the amplitude to 22 centimeters up and 22 centimeters down from neutral and start the device with an initial cycle period at 2, 500 milliseconds for five minutes, 2, 000 milliseconds for five minutes, and 1, 500 milliseconds for five minutes. Then set the device to 1, 000 milliseconds for two hours before slowing the device in reverse using periods of 1, 500 milliseconds for five minutes, 2, 000 milliseconds for five minutes, and 2, 500 milliseconds for five minutes.
For a Ferris-wheel rotation procedure, place the plexiglass container on a wooden bench and place the rats into the container with the long axis of their bodies perpendicular to the horizontal rotation rod of the Ferris-wheel. After closing the box securely, place a second set of rats with a similar mass into a second plexiglass container with the long axis of the body perpendicular to the horizontal rotation rod on the second arm of the Ferris-wheel rotation device. After securely closing the box, start the Ferris-wheel rotation in a clockwise direction at 16 degrees per second squared to reach an angular velocity of 120 degrees per second.
When the Ferris-wheel has reached peak velocity, begin to decelerate at 48 degrees per second squared to reach zero degrees per second. After a one-second pause, have the container continue to rotate in the counterclockwise direction at 16 degrees per second squared to reach an angular velocity of 120 degrees per second before decelerating at 48 degrees second squared to reach zero degrees per second. Then continue the clockwise-counterclockwise rotation for two hour per session for approximately 720 rotations.
For balance beam training, first place two wooden 0.75 meter high stools into the experimental field approximately 110 centimeters apart. Place a plastic black 15 by 15 by eight centimeter box on the finish tool and place a narrow 2.5 by 130 centimeter wooden beam between the two stools leaving a 100 centimeter distance between the stool edges from the start stool to the finish stool. Place a lamp at the start stool and turn on the lamp.
Then turn off the room light so that the room is as dark as possible and introduce the first rat to the beam in the lighted corner. If the rat does not cross of its volition, gently prompt the animal to cross the beam. After three consecutive days of training, place the trained rat onto the start stool near the light and immediately start a stopwatch.
The rat should cross the balance beam rapidly and enter the black box on the finish stool. Stop the stopwatch as soon as the animal's nose enters the dark box on the finish stool. For defecation counting after motion treatment, transfer the rats from the plexiglass container into individual 40 by 40 by 45 centimeter open field boxes and count the number of feces pellets in the plexiglass box attributed to each rodent.
For an open field behavior test, use an infrared video camera to record the animals for three minutes and use the resulting video footage to determine the total distance traveled for each animal. Here, representative balance beam results of the time taken to traverse the balance beam after three consecutive days of training are shown. Elevator vertical motion, changes in the Hertz vertical motion, and Ferris-wheel rotation significantly increased defecation.
In addition, elevator vertical motion, changes in the Hertz vertical motion, and Ferris-wheel rotation significantly decreased the open field distance traveled. The balance beam training is critical as the rats must be motivated and confident when crossing the beam. Otherwise, their balance will not be measured during the evaluation period.
Following the behavioral procedures, the effects of different drugs of interest on autonomic functioning and cell signaling can be assessed using these vestibular stimulation assays.
