Procedures

General Information for All Procedures: The PhenoMaster automated home cage setup has been previously described by Chevessier et al. 2015.

Procedure 1: Monitoring system for physical activity and energy expenditure

Equipment, software, and supplies

• Standard home cage environment, IVC Sealsafe Plus cage (Tecniplast S.p.A., GM500, Buguggiate, Varese, Italy)
• Automated phenotyping home cage system, 8-place (TSE Systems, PhenoMaster, Bad Homburg, Germany)
• PhenoMaster software (TSE Systems, Version 4.8.9, Bad Homburg, Germany)

Environmental Conditions

Holding Area and/or Sample Collection Area

Facility: Holding room at the preclinical research center of the Universitätsklinikum Erlangen

Acclimation Period: 7 days

Description of Holding Enclosure: Mice are transferred to the holding room at least 7 days before the start of the experiment and kept in standard home cage environments

Max # Animals Per Enclosure: 5

Temperature (°C): 22 +/- 2

Humidity: 55 +/- 10

Testing Area

Facility: Testing room with PhenoMaster automated home cage phenotyping setup

Acclimation Period: First day of two day experiment

Light Source: Standard neon lights

Light Intensity: 1000 lux in the room, and 400 lux in the cages

Temperature (°C): 22 +/- 2

Humidity: 55 +/- 10

# Mice Tested at the Same Time: 8

Notes: Disturbance during the testing period are kept to a minimum, entries into the room are recorded and the technician entered only once a day to control the PhenoMaster setup, inspect the health of the animals and refill water bottles/feeders, if necessary

Steps

1. Test mice are monitored in the PhenoMaster automated home cage phenotyping system which is used to assess the circadian pattern of locomotor activity, ingestion behavior, and indirect calorimetric parameters in a standard home cage environment. The experiment lasts for 2 days in total, and started latest at 4 pm with one scotophase, on the first day. This is done in order to acclimatize test mice to the newly provided home cage within the PhenoMaster setup. Data is recorded on the second day (light and dark cycles) and is used for analysis. Data are recorded for up to 8 animals in parallel. The body weight (g) and age (days) of all mice are recorded in the first day of the experiment.
2. Spontaneous ambulatory and fine movements (XY plane) and rearing events (Z plane) are detected by infrared light beam frames. Two dedicated weighing sensors per cage register ad libitum water and food consumption.
3. Continuous mode calorimetry, via one O₂/CO₂ gas sensor pair per cage, allows the simultaneous measurement of O₂ and CO₂ concentrations and the calculation of O₂ consumption, CO₂ production, respiratory exchange ratio (RER = VCO₂ [mL/h/kgBW] / VO₂ [mL/h/kgBW]) and heat (H = [kcal/h/kgBW]) (performed by TSE PhenoMaster Software). All gas sensor pairs are calibrated with calibration gas mixtures (CO₂ 0.05%, O₂ 20.895%, in N₂; CO₂ 0.950%, O₂ 20.00%, in N₂) once a week.
4. After each experimental run, the 8 mice are returned to their original social groups, and transferred to the regular holding room.