A Chronic Immobilization Stress Protocol for Inducing Depression-Like Behavior in Mice

Begin by setting the room to light at 200 lux using a digital lux meter. House the mouse in a separate cage at least a week before testing, and place the mouse in the testing room for at least 30 minutes before the experiment. Gently hold the mouse tail to avoid tensing the mouse, and then carefully place it on a rough surface.

Cover the restrainer with a small white towel and then gently place the mouse at the opening of the restrainer, so that the mouse enters the restrainer voluntarily. Next, place the closure to restrain the mouse as tight as possible, being careful to avoid damage to the body, such as tail, feet, and testicles. Lastly, during the exposure to the restrainer, measure body weight and food intake every 48 hours.

Prior to testing, habituate the mice to the presence of two drinking bottles, one containing 0.1 molar sucrose and the other containing plain water for 48 hours. Switch the positions of the two bottles after 24 hours to reduce any confounding produced by a side bias. On the third day deprive the mice of water for 24 hours.

Then on the day of the experiment expose the mice to two drinking bottles for six hours. After three hours, switch the position of the water bottles. Record the volume in milliliters of sucrose solution and water consumed, and then calculate the animal's affinity to sucrose.

Finally, calculate sucrose preference as a percentage of the volume of sucrose consumption over the total fluid consumption during the test. Begin by bringing the chronic restraint stress induced mice into the testing room at least 30 minutes before beginning the tail suspension test. Set the room light to dim conditions at 50 lux.

Next, place the camera about 40 centimeters from the mouse. Suspend the mouse firmly from the horizontal bar using adhesive tape. After the mouse is positioned in the middle of the suspension box start recording, and observe the behavioral alterations continuously for six minutes.

If the mouse attempts to climb its tail use a stick or climb stopper to prevent it from doing so. At the end of the experiment move the mouse to its home cage and carefully remove the tape from its tail. Finally, use video tracking software to analyze the accumulated time of immobile periods.

Results indicate decrease in body weight for the CIS group and significantly less food intake at day four. Compared to controls, a decrease was shown for sucrose preference in the CIS group, while there was a significant increase in immobility time and corticosterone. Further, a glutamine supplemented diet ameliorates depressive like behaviors, as shown for body weight, food intake, sucrose preference, immobility, and corticosterone.