Getting to Compliance in Forced Exercise in Rodents: A Critical Standard to Evaluate Exercise Impact in Aging-related Disorders and Disease

The overall goal of the following experiment is to obtain maximum compliance in rodent test subjects to exercise on a running treadmill without the use of any form of negative reinforcement. This is achieved by first obtaining baseline measurements of the parameters that are intended to be evaluated in this case, locomotor activity in an open field. Following the implementation of an exercise regimen as a second step, the rodents are acclimated to obtain maximum compliance to the exercise regimen.

This includes familiarity with the treadmill environment, learning to run on a slow moving treadmill and ensuring that a backstop is in place on the treadmill lanes. So the test subjects are always segregated from deactivated shock coils. Next, the exercise regimen is employed for 12 consecutive days at 30 minutes per day.

The non-exercise group sits on a stationary treadmill for the same duration. The results show this exercise regimen produces a significant difference in weight loss of rats as compared to their non-exercise counterparts demonstrating its use to independently evaluate exercise impact upon measures previously determined at baseline. The main advantage of this technique over existing methods is that maximum compliance to forced treadmill exercise can be obtained in rodent test subjects and in particular age rodents without the use of any form of negative reinforcement.

In order to best understand the neurobiological impact of exercise on CNS function, it's important to match the duration, frequency, and intensity of exercise with behavioral and neurobiological outcomes. It is the goal of this protocol to help investigators achieve these aims. To establish baseline locomotor capabilities, each subject spontaneous locomotor activity is assessed for 60 minutes per day for five consecutive days.

Conduct locomotor activity sessions in automated open field locomotor activity chambers consisted of a plexiglass box with a grid of infrared beams mounted horizontally and vertically. As suggested in the product manual at the beginning of each day of the locomotor activity assessments load and set up the computer and software program. The program communicates with the open field locomotor activity chambers in order to record the number of beam breaks during each session.

Next, at approximately two cups of pine chip bedding to the floor of the locomotor chamber prior to each session, transport the subjects from the animal colony to the locomotor activity chambers in their home cages immediately prior to their locomotor activity session. Load the subjects into their assigned locomotor chamber under a red light. Start the 60 minute session on the computer once they have been placed into the chamber.

Upon completion of the locomotor activity session, remove the subjects from the chamber and return them to their home cage Under red light, immediately return the subjects back to the animal colony. Then remove the bedding from the chamber and clean the chamber walls and floor with 50%ethanol after each session from the data generated by the software used to measure locomotor activity. Determine each animal's baseline locomotor activity with five locomotor parameters from these locomotor parameters.

Determine exercise and non-exercise groups such that an equal range of locomotor activity is represented in both groups. Pair rats together based on similar movement capabilities and assign one rat to the exercise group and one rat to the non-exercise group. Conduct all treadmill exercise related procedures during the animals active cycle and one to two hours prior to the light dark cycle.

Change on a motorized rodent treadmill with lanes that are separated by clear plexiglass walls. Insert a plexiglass backstop designed to fit the treadmill so that subjects can continuously exercise without coming into contact with the electrical shock coils to prevent the backstop from sliding. During the training sessions, hold the backstop in place with a 1.5 inch sea clamp transport subjects in their home cage to the treadmill immediately prior to their treadmill exercise training session.

When the treadmill is stationary, load rats onto the treadmill in their assigned lane by sliding up the backstop just enough that subjects can maneuver under the backstop. Slide the backstop back down to the resting position. Once the subject is situated on the treadmill belt.

During the first phase of acclimation, place each rat on one of the four lanes of the stationary treadmill for five to 10 minutes per day for three consecutive days. Do not place rats anywhere on the treadmill except the stationary belt. At this stage, during the second phase of acclimation training, place each rat on one lane of the stationary treadmill in front of the backstop.

When all subjects are in place, turn the treadmill on to a rate of five meters per minute. As the belt of the treadmill begins to roll, ensure that all rats are walking in the forward direction, assist rats as necessary by orienting or gently prodding them until they are moving forward. Continue acclimation of the rats as described in the text protocol.

Upon completion of the treadmill acclimation session. Remove subjects from the treadmill in the same manner used to load them immediately. Return subjects to their home cage and transport them back to the animal colony.

Wipe down each individual lane, including the walls and treadmill belt with 50%ethanol. After each individual treadmill exercise training session, ascertain the exercise capabilities of each rat across the exercise sessions as described in the text protocol accompanying this video. Begin the exercise training sessions the day following the last acclimation training session and continue exercising rats for 12 consecutive days following the same setup procedures used during acclimation training.

At the start of each session, turn on the treadmill to a warmup speed that is lower than the training speed to be maintained throughout the remainder of the exercise training session. Continue with this warmup speed for five to 10 minutes at the beginning of the session. Then adjust the speed to between nine and 11 meters per minute for the remainder of the training session.

Train subjects an average of 30 minutes each session for the entire 12 days of exercise training. Conduct the exercise and non-exercise sessions separately. Place the non-exercise rats on the stationary treadmill for the same duration that their exercise cohort trained during each session.

Representative results of baseline locomotor activity parameters demonstrate no significant differences in any of the parameters between exercise and non-exercise test subjects prior to treadmill acclimation training. These parameters include total distance, baseline, horizontal activity, baseline movement number, baseline movement time and baseline movement speed. Average treadmill exercise scores following acclimation and exercise training are shown as an indicator of compliance to treadmill exercise during each session.

Scores range from one to four with four indicating 100%compliance for an exercise training session. As shown here, there is no significant difference in mean treadmill exercise scores between exercise and non-exercise rats. During treadmill acclimation exercise, rats were compliant through two rounds of treadmill exercise as indicated by mean treadmill exercise scores of 3.80.

No significant difference in mean treadmill. Exercise scores were observed between the two rounds of exercise, significant differences in weight loss and percent decrease in body weight between exercise and non-exercise. Rats were observed during the two rounds of treadmill exercise, but not during baseline or acclimation periods demonstrating the impact of exercise on body weight With adherence to the recommended treadmill, acclimation procedures and assessment of baseline performance measures, the investigator will be able to evaluate the impact of exercise at a given intensity, duration, and frequency upon the neurobiological underpinnings and behavioral outcomes associated with exercise.

The key feature of this protocol is that age rodents can be trained to exercise with nearly a hundred percent compliance without the use of foot shock at any point in this procedure. This eliminates the need for additional test subjects to replace those that are non-compliant. It also prevents potential confounds that foot shot can produce on neurobiological function.

We expect that this protocol should be useful for investigators to simply evaluate the impact of exercise upon behavior and neurobiological function.