Loos2 project protocol

Sheltering behavior and locomotor activity in males of 11 inbred strains of mice and within-strain variation of 8 of those strains   (2016)

Loos M, Koopmans B
With: Aarts E, Maroteaux G, van der Sluis S, Verhage M, Smit AB, Neuro-BSIK Mouse Phenomics Consortium

Project protocol - Contents

Workflow and sampling

Data collected*
Three days of testing in a home-cage environment to capture spontaneous behavior measures PhenoTyper
Measures of locomotor activity and sheltering

*Supplementary data are available for this project. See Loos2 downloads.

Equipment and supplies

  • PhenoTyper model 3000 (Noldus Information Technology, Wageningen, The NETHERLANDS)
    • The cages (L=30, W=30, H=35 cm) are made of transparent Perspex walls with an opaque Perspex floor covered with cellulose-based bedding.
    • A feeding station and a water bottle are attached onto two adjacent walls.
    • A triangular-shaped shelter compartment (H=10 cm) made of non-transparent material has two entrances fixed in the corner of the opposite two walls.
    • The top unit of each cage contains an array of infrared LEDs and an infrared-sensitive video camera used for video-tracking.
  • EthoVision software (EthoVision HTP, based on EthoVision XT 4.1, Noldus Information Technology, Wageningen, The NETHERLANDS
  • AHCODA analysis software (Synaptologics BV, Amsterdam, The NETHERLANDS


Figure 1. Schematic of home cage.

Procedure: Automated home-cage observation

    1. Mice are tested in the PhenoTyper cage to assess spontaneous behavior for 3 consecutive days.
    2. Mice are introduced to the test cages in the second half of the subjective light phase (14:00h-16:00h); video tracking starts at the onset of the first subjective dark phase (19:00h).
    3. X-Y coordinates of the center of gravity of each mouse (sampled at a resolution of 15 coordinates per second) are acquired and smoothed using EthoVision software and processed to generate behavioral parameters using AHCODA analysis software.
    4. Move and arrest segments are separated by repeated running medians smoothing of X-Y coordinates (for details see Hen et al.). See Figure 2 for illustrated definitions of of endpoints.
    5. Smoothing settings are chosen such that move segments represent gross movements of the center of gravity, e.g, locomotor activity or rearing; arrest segments reflect complete inactivity or minute movements of the center of gravity, e.g., grooming or eating; and shelter segments are recorded if the center of gravity of a mouse disappeared in the 2-cm zone drawn immediately in front of the shelter entrance.
    6. A shelter segment is ended if the center of gravity is detected continuously for at least 7 samples (0.5 s).
    7. Three additional zones are digitally defined: a Feeding zone, a Spout zone, and an OnShelter zone.
    8. Mice which spend little time in the shelter (<60% of time in the shelter during light phase of days 2 and 3) in combination with being highly inactive outside the shelter (cumulative movement less than 2 cm per 5 min for >25% of time outside during light phase of days 2 and 3) are classified as sleeping outside the shelter and are excluded from the analyses.
    9. Elements of behavior are identified by mouse-determined thresholds.
    10. Short movements (turning or rearing against the wall) and long movements (when mice travel from one location in the cage to the next) are identified.
    11. Mice frequently visit the shelter for a few seconds (short shelter visits) during bouts of activity; long shelter visits indicate resting or sleeping.
    12. To improve the detection of spontaneous behavior in the home-cage, existing analysis methods are adapted to segment continuous behavioral observations into distinguishable behavioral elements (for review, see Benjamini et al). See Loos et al. for details.


    Figure 2. Segmentation of sheltering behavior and activity into elements. A representative track of ~17 min for a C57BL/6J mouse, dissected into elements by individually determined thresholds.

Procedure: Analysis of spontaneous behavior

    1. Activity bouts are defined (start with a long movement and stop when a long arrest segment is encountered, or a shelter visit exceeds the brief shelter visit threshold). Characteristics of activity bouts are binned in 12h time bins, and cumulative and mean duration and/or frequencies are calculated.
    2. A habituation index for a given parameter is calculated by taking the ratio of a 12h time bin on day 3 over day 1.
    3. A DarkLight index is calculated from the 12h time bin values on the third day: (dark value/dark value + light value)).
    4. Activity patterns are analyzed in terms of the change in the proportion of time active in the hours preceding and following the shift in light phase.
    5. The last and first 10 min of each dark and light phase are not included in parameters.

Procedure: Analysis of within-strain variability

    1. Principal component analysis (PCA) is performed with Varimax rotation on the data of individual mice for all 115 behavioral parameters after subtraction of strain means to focus on within-strain variability (missing data replaced by strain means). Note that three strains were dropped from the analysis due to low sample size, leaving eight strains.
    2. Subject's scores on PC are estimated using regression.
    3. ANOVA and PCA are performed with SPSS v 20.0.

    PCA identified 22 orthogonal PCs of within-strain variability across the entire dataset.

Data collected by investigator

  • activity bouts
  • habituation
  • dark-light index
  • light-dark phase transitions
  • within-strain variability


Benjamini Y, Lipkind D, Horev G, Fonio E, Kafkafi N et al. 2010. Ten ways to improve the quality of descriptions of whole-animal movement. Neurosci Biobehav Rev 34:1351-1365.

Hen I, Sakov A, Kafkafi N, Golani I, Benjamini Y. 2004 The dynamics of spatial behavior: how can robust smoothing techniques help? J Neurosci Methods 133: 161-172.