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.

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.