Elevated Plus Maze Test Combined with Video Tracking Software to Investigate the Anxiolytic Effect of Exogenous Ketogenic Supplements

Here, we present a protocol to investigate changes in the anxiety level of rodent animal models. The elevated plus maze (EPM) test, used together with a video tracking software, provides a reliable method to document the effect of various potential anxiolytic treatments in preclinical laboratory scenarios.

The elevated plus maze is a method to document the effect of various potentially anxiolytic treatments on laboratory rodent models in a more humane way compared to other methods. The main advantage of this method is that is relies on the rodent's instinctive proclivity to both dark and closed spaces, in addition to the unconditioned fear of heights and avoidance of open spaces. Collin Park, a collaborator from my laboratory, will help demonstrating the procedure.

Several days before testing, handle the animals by picking them by their torso and holding them for a minute to acclimate them to the experimenter. Then, measure the body weight of the animals before starting any treatments to determine the dosage calculation for treatment. Next, familiarize the animals to the intragastric gavage method using water by gavage for five days before ketone supplementation.

For the anxiety assay, use the elevated plus maze or EPM Apparatus, a plus shaped apparatus with four arms with two open and two closed. Light up the EPM Apparatus by using indirect lighting from the ceiling instead of directly illuminating it. Ensure all four arms are similarly illuminated.

Plug the installation key of the movement tracking software into a USB 2.0 port and install the software. Then, fix the camera above the experimental area, and ensure that it will stay immobile for the duration of the experiment. Finally, set up a new experiment in the movement tracking software system by using the instruction manual.

Place the animal into the experimental area. Then, start the data acquisition process by pressing the start button on the time control panel. In the testing room collect the EPM data in two ways, manually and by using the software.

The blinded observers are separated by a curtain. Wait until the ene of the tracking process recording or press the stop button on the time control panel. Then, remove the animal from the experimental area.

Finally, to access the analysis tool press the analysis button in the experimentation assistant bar. Select trials and time intervals to analyze and generate a report. Begin by transferring the rats in their home cage to the experimental room 30 minutes prior to beginning the experiment.

Place a rat at the intersection of the four arms of the EPM facing the open arm opposite of the experimenter. Run the video tracking software as well as manually record the behavior of the animal for five minutes. Then, if the animal falls off the EPM, pick it up and place it back on the same point of the EPM where it fell off.

Exclude the behavioral data of this animal from the analysis. At the end of the five minute test, stop the video tracking software. Remove the animal from the EPM, and place it back on its home cage.

Finally, clean the EPM with a disinfecting detergent followed by tap water. Dry the apparatus with paper towels. After chronic feeding, rats in the KSMCT group spent significantly more time in the open arms compared to the control group.

The KS group spent significantly more time in the center compared to the control group. And less time is spent in closed arms in the LKE, KS, KSMCT groups compared to control. Rats in the KS and KSMCT groups traveled significantly longer distances in the open arms.

While the rats in the LKE, KS, and KSMCT groups showed significantly less distance traveled in the closed arms compared to the control group. Further, after seven days of oral gavage the time spent in the open arms was greater in the KE group but decreased in the center for the KE, KS, and KSMCT groups. The number of entries to the closed arms was also significantly lower after seven days of administration in the KE and KS groups.

It is very important to place all rats on the equipment in a consistent manor at the intersection facing away from the experimenter. EPM assay on rodents is a suitable and sensitive method to investigate the effect of different treatments that influence brain areas involved in the anxiolytic effect and mechanisms of action.