Here we present the unpredictable chronic mild stress protocol in mice. This protocol induces a long-term depressive-like phenotype and enables to assess the efficacy of putative antidepressants in reversing the behavioral and neuromolecular depressive-like deficits.
The main advantage of this technique is that it adequately translates major neurobiological and behavioral features implicated in the pathogenesis, etiology, and treatment of depression. This method can help answer key questions in understanding the pathophysiology of anxiety and depression and promote the development of novel pharmacological treatments. The implication of this technique extend for therapy of depression, because it enables screenings for potential antidepressants while resembling the protracted course of remission evident in human patients.
Begin by filling home cages with fresh sawdust, and add a piece of cotton wool for enrichment. House animals in the home cage for an acclamation period of one week, and keep a consistent 12-hour light/dark cycle. Allow ad libitum access to rodent chow and water.
Design a four-week stressor regimen in which each of the seven stressors is utilized once a week, on a different day each week. After one week of acclamation, initiate application of the stressors on the four-week-old mice in the UCMS room. For the first stressor, place mice together with their home-cage counterparts in an empty cage.
Fill the empty cage with water kept at 24 plus-or-minus-one degrees Celsius to a depth of one centimeter, and keep mice in the wet cage for four hours. Then transfer each mouse to a separate individual transient drying cage with a heat lamp above it, a heating pad under it, and paper towel bedding. Place a thermometer in the transient cage to verify the temperature does not exceed 37 degrees Celsius.
Keep each mouse in the transient cage until it is dry and looks invigorated, and then return mice to the home cage with the same counterparts. For dampened sawdust, poor water kept at 24 plus-or-minus-one degrees Celsius into the home cage until the sawdust is moderately dampened. After four hours, dry the mice in transient cages.
Then place the mice with home-cage counterparts in a sterile cage with fresh sawdust. For the next stressor, tilt the cage at 45 degrees against the wall for four hours. For Empty Cage, transfer all five mice along with their specific home-cage counterparts from the home cage to an empty cage for four hours.
For the fifth stressor, transfer mice along with their specific home-cage counterparts from the home cage to a cage which was housed by a different group of mice for a period of at least three days prior to stressor application. Keep mice in the unfamiliar cage for four hours. For the sixth stressor, place each mouse separately in a clean mouse restrainer for four hours.
After that, return mice to the home cage with the same counterparts. For the last stressor, transfer mice in their home cage with their specific counterparts to the UCMS room. Keep the light on for 24 consecutive hours.
Finally, after stressor application, return the cages of the UCMS group from the UCMS room to the housing room. During the four weeks of stress exposure, keep the naive group in their home cages located in the housing room. On the day following cessation of the UCMS protocol, start administration of putative therapeutic pharmacological agents.
After the treatment phase, remove each mouse from the home cage and place it individually in a cage filled with fresh sawdust and a piece of cotton wool for enrichment. Then prepare two bottles, one with distilled water, and another with 2%sucrose solution. Weigh the two bottles and set them at both ends of the cage lid and place rodent chow between the two bottles to allow mice ad libitum access to both solutions and food for a period of 24, 48, 72, or 144 hours.
After 12 hours, or once a day if the test duration exceeds 24 hours, weigh the bottles to estimate consumption from each bottle and switch the positions of the nozzles to counterbalance the possibility that the results were confounded by position preference. Finally, weigh the bottles each day to estimate consumption from each bottle. The UCMS group demonstrated diminished sucrose preference compared to the naive group, suggesting that the UCMS protocol was effective in inducing anhedonia.
Furthermore, the UCMS group demonstrated diminished hippocampal BDNF levels compared to the naive group, suggesting that the UCMS protocol led to the diminution in hippocampal BDNF levels as evident in human depression. Results indicated that the UCMS saline group demonstrated a significant decrease in sucrose preference compared to the naive saline group as well as compared to both the UCMS escitalopram and the UCMS NHT groups, suggesting both escitalopram and NHT normalized the UCMS-induced anhedonia. In addition, the UCMS saline group demonstrated decreased hippocampal BDNF levels compared to the naive saline group as well as compared to both the UCMS escitalopram and the UCMS NHT groups, suggesting that both escitalopram and NHT normalized the UCMS-induced reduction in BDNF levels in the hippocampus.
Following this procedure, other methods like tail suspension test, forced swim test, and elevated plus-maze can be performed. These tests are used to assess behavioral despair as well as exploratory and anxiety like behavior.
