An Unpredictable Chronic Mild Stress Protocol for Instigating Depressive Symptoms, Behavioral Changes and Negative Health Outcomes in Rodents

All procedures described below have been reviewed and approved by the Institutional Animal Care and Use Committee at the West Virginia University Health Sciences Center.

1. Selection of Animal Model 

NOTE: Most commonly used species in the UCMS model include Sprague Dawley, and Wistar rats and the BALB/cJ mouse; other murine models have shown limited efficacy (DBA/2, C57BL/6).  However, careful consideration should be given to the particular mouse or rat strain of choice, as the genetic background and susceptibility to environmental stressors vary widely among species and have been shown to be critical factors affecting the outcomes of the study^11,14,15,19.

1. Use animals of the appropriate age (e.g., if the desired endpoint is to be evaluated in a 17-week old rat, then the rats must start the UCMS protocol at ~9 weeks of age) as this protocol takes at least 7-9 weeks to fully induce depressive symptoms.
2. Single-house animals undergoing UCMS in IACUC-approved cages and facilities, with food and standard chow available ad libitum (unless otherwise required by specific protocol).
3. Take special care to ensure controls are housed in a quiet room with minimal activity to reduce effects of any possible unknown environmental stressors.

2. Unpredictable Chronic Mild Stress Protocol 

NOTE: Animals undergoing UCMS are transported daily (>5 days per week) to a clean room used for UCMS manipulations. Animals are exposed to 1 or 2 of the stressors listed below each day. Stressors are performed on a randomized schedule, and each stress is administered for a minimum of 3-4 hr (see Table 1).

1. Dampen the bedding by pouring 10-20 oz. of clean water into each standard cage. Adjust the amount of water accordingly to the size of the cage in order to fully dampen the bedding but not to cause pooling of water. Reside the animals in damp cage for 3-4 hr.
2. Remove bedding from each cage for 3-4 hr, after which animals are transitioned into another stressor requiring an empty cage (shallow bath, cage tilt) or placed into a clean cage with fresh bedding.
3. Tilt cages to approximately 45° (without bedding) for 3-4 hr. Cages can be tilted with a sturdy object that will remain in place as the animal moves around in the cage.
4. Alter normal 12 hr/12 hr light/dark cycles into successions of 30 min periods lasting for 8 hr. After, return animals to the normal cycle.
5. Transfer each animal from its home cage to that of the cage of a neighboring animal that has been removed for 3 hr.
6. Remove all bedding from each cage and add water to a depth of ~0.25 inches for mice or ~0.5 inches for rats for approximately 4 hr. Use water that is warmer than RT (~30 °C) to minimize hypothermia potential. Briefly dry animals with a soft towel at the conclusion of the water exposure prior to placement into clean cages.
7. Expose animals to predator smells/sounds by adding randomly distributed sample tufts of fur or urine (10-20 ml) from a natural predator species into the cage for 3-4 hr. Alternatively, play growling or predatory noises from natural predator species in close proximity to cages for 3 hr.
8. At the end of each daily stress period, place all animals into clean cages and return them to the housing facility.

3. Animal Monitoring/Grooming Scores

1. Inspect each animal daily for the development of wounds or sores that may require veterinary treatment. Animals that develop mild injuries may continue in the UCMS protocol on the consent of the veterinarian.
   1. Two investigators trained in evaluating coat grooming inspect each animal and assign a weekly coat score on a scale of 0-1. This score is determined by assigning individual scores (0- clean, 1-dirty) to each of 8 body regions- head, neck, dorsal coat, ventral coat, tail, forelimbs, hind limbs, and genital region. The same investigators are to perform this task throughout the UCMS protocol to maintain consistency in assigning coat scores.
   2. Any animal that experiences a sharp reduction in body weight (i.e., >15%) should be reported to the veterinary staff and must be removed from the protocol.

4. Behavioral Testing and Outcome Measurements 

NOTE: Several methods of assessment can be used to quantify behavioral changes from the UCMS protocol in rodents.

1. Cumulative Coat Grooming Score.
   1. As described above, a weekly coat grooming score is assigned for each animal.
2. Sucrose Splash Test:
   1. Place the animal in a clean cage lined with an cotton pad or towel to prevent bedding from adhering to its coat. Spray a 10% sucrose solution onto the head and dorsal coat of each animal and record total grooming behavior for 5 min.
   2. Grooming activity (defined as cleaning of the fur by licking or scratching) is measured by latency (idle time between the first spray and initiation of grooming) and frequency (number of times grooming a particular body part).
3. Sucrose Preference Test:
   1. Habituate animals to drink 1% sucrose solution (w/v) 72 hr before the test and then provide exposure to two bottles (1% sucrose solution versus tap water in different bottles). After habituation, provide animals with ad libitum access to sucrose solution and tap water for 3 hr. After 3 hr record the consumption volumes of sucrose solution and tap water and calculate sucrose preference as: [(sucrose consumption)/(water consumption + sucrose consumption)]×100.
4. Tail Suspension Test (Mice Only):
   1. Suspend mice by the tail from a stable rod (20 cm from the floor) using adhesive tape (2 cm from tip of tail). Record immobility time over 5 min. Mice are considered immobile only when they hang passively and completely motionless.
5. The Forced Swim Test:
   1. Place animals in a container (~15-20 cm diameter for mice; ~55-60 cm diameter for rats) filled with water at a temperature of 26 ± 0.5 °C. Record the latency to the first bout of mobility and the time spent immobile during the 5-min testing period. At the end of a test, place the wet animal in a holding cage on a heating pad (30–35 °C) with normal bedding covered by an absorbent paper towel until dry.
6. Elevated Plus Maze:
   1. Allow each animal to explore the maze freely for 5 min. Record total entries into each arm, and total time spent in each arm, according to a priori criteria²⁰.
7. Locomotor Activity:
   1. Record activity levels, defined as overall movement, fine movements, rearing movements, and inactivity, for both acute (30 min) and/or chronic (12 hr) time periods. Do this using the home cage or a novel environment, depending on the individual environment (although consistency should be maintained) in real time by an observer or post-hoc by replaying and analyzing camera footage.

5. Expansion/Variation of UCMS Protocol

1. Administer appropriate therapeutic agents or challenges to study their ability to either blunt the development of UCMS-induced outcomes (if initiated early) or to reverse severity of established UCMS-induced outcomes (if initiated late). This is dependent on the individual laboratory or protocol¹¹.

6. Final Outcome Assessments

1. Following the conclusion of UCMS protocols and behavioral testing, utilize animals in subsequent experiments in a manner that addresses the specific hypotheses, aims or goals of the individual laboratory.  
   NOTE: As examples, this can include cardiovascular, inflammatory or endocrine responses to chronic stress. Alternatively, therapeutic interventions can be introduced at this point to evaluate their effectiveness and mechanistic underpinnings.