Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Open field test
• Procedure: Light-dark test
• Procedure: Stress-induced hyperthermia test
• Procedure: Forced swim test
• Procedure: Stress response test (corticosterone levels)
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	At 5-6 wks of age, dams of selected Collaborative Cross strains are subjected to either a control diet or a specialty diet (see Animal Documentation) for 5 wks	-	-
2	After 5 wks on their respective diets, breeding cages are set up to produce F1 hybrids; dams are continually administered the experimental diets during gestation and through weaning	-	-
3	Body weights at weaning (day 21) and just before testing (~day 60)	Scales	Body weights
4	Open field test	Open field arena	Number of fecal boli, distance traveled, rearing activity, time in center of arena
5	Light-dark test	Light-dark box	Number of fecal boli, number of transitions (dark-light), distance traveled, time in light side
6	Stress-induced hyperthermia	Digital thermometer	Basal body temperature and temperature 10 min after basal temperature taken
7	Forced swim test	Cylinder filled with water	Number of fecal boli, time spent immobile
8	Stress response test	Restrainer	Corticosterone levels: before and after restraint

Equipment and supplies

• Scales
• Open field apparatus (ENV-515-16, Med Associates, St. Albans, Vermont)
  • 43.2 × 43.2 × 33 cm arena
  • White Plexiglass floor and clear Plexiglas walls
  • Infrared detection beams at 2.54 cm intervals on the x, y and z axes
  • Enclosed in a sound-attenuating chamber (73.5 × 59 × 59 cm)
  • Fitted with 2 overhead light fixtures containing 28-V lamps
  • Center is defined as the 22.86 cm² central part of the arena
• OF software (Activity Monitor 5.1, Med Associates St. Albans, VT)
• Light-dark box (Versamax420 Animal Activity Monitoring System, AccuScan Instruments Inc., Columbus, OH)
  • 42 × 42 × 30 cm arena
  • White Plexiglass floor and clear Plexiglas walls
  • Surrounded by 16 photobeams along each side
  • The black Plexiglas box (40 × 21 × 13 cm) occupies one-half of the arena and has a 10 × 3 cm opening to the light side and holes on all 4 sides that allow detection of movement by the photobeams
• LD software (VersaMap version 1.7, AccuScan Instruments, Inc)
• Digital rectal thermometer (TH-5 Thermalert Monitoring Thermometer with RET-3 rectal probe, Physitemp Instruments, Clifton, NJ)
• Forced swim apparatus
  • Glass-polycarbonate cylinder (46 cm tall × 21 cm in diameter) filled with water to a depth of 15 cm and maintained at a temperature of 25°C to 28°C
• FST software (Ethovision 7.0 automated tracking software, Noldus, Leesburg, VA)
• Broome-Style restraint tube (Plas Labs, Inc., Lansing, MI)

Reagents and solutions

• Disinfectant
• Diets (see Animal Documentation)
• Competitive radioimmunoassay (RIA) (MP Biomedicals, Santa Ana, California)

Procedure: Open field test

1. Mice are placed in the OF arena for 10 min and scored for total distance traveled (cm), number of vertical movements (rearing) and percent time spent in the center of the arena.
2. Data are analyzed post-session in 2-min bins using commercially available software.

Procedure: Light-dark test

1. Mice are placed in the lighted area immediately adjacent to and facing the entry to the dark enclosure and left to freely investigate the apparatus for 10 min. The amount of time (seconds), number of transitions, distance moved (cm) and percent time in the dark and light zone.
2. Data are analyzed post-session in 2-min bins using commercially available software.

Procedure: Stress-induced hyperthermia test

1. Mice are individually removed from the home cage and the initial temperature (T1) is measured by insertion of a lubricated digital thermometer probe 1 to 1.5 cm into the rectum for approximately 10 seconds.
2. The animal is immediately returned to the home cage and 10 minutes later, the temperature measurement is repeated (T2).
3. The difference in body temperature between T2 and T1 is calculated as the change in temperature (ΔT).

Procedure: Forced swim test

1. Mice are tested for 6 min.
2. Percent immobility during the last 4 min of the test period is recorded by video and analyzed by software.
3. Immobility is defined as the mouse making no movements other than those needed to stay afloat.
4. Mice are monitored continuously and removed from the apparatus if they are unable to keep their nose or heads above water for more than 30 seconds.

Procedure: Stress response test (corticosterone levels)

1. Restraint is used to elicit a stress response that is quantified by measurement of corticosterone (CORT) levels in the serum.
2. A retroorbital blood sample is taken from unanesthetized mice to assess basal CORT levels.
3. Then mice are immediately placed into a restraint tube for 10 min.
4. Immediately upon removal from the restrainer, a second unanesthetized retro-orbital eye bleed is performed to assess stress-induced CORT levels.
5. Whole blood is centrifuged to isolate serum and CORT levels are measured using a competitive radioimmunoassay (RIA) using the manufacturer’s
   protocol.

Data collected by investigator

• Body weight
  
• Open field test
  • Number of fecal boli
  • Distance traveled
  • Vertical activity
  • Time in center
• Light-dark test
  • Number of fecal boli
  • Number of transitions between light and dark compartments
  • Distance traveled in both compartments
  • Distance traveled total
  • Time in light compartment
• Stress-induced hyperthermia test
  • Basal temperature
  • Temperature 10 min after basal temperature taken
  • Change in temperature
• Forced swim test
  • Number of fecal boli
  • Time spent immobile
• Stress response test
  • Corticosterone level: basal
  • Corticosterone level: after restraint
  • Change in corticosterone level