Knowledge Base

Problem

A user setting up a three-chamber social interaction test is unsure how to position the restraint cage within the apparatus and how to structure the experimental procedure in EthoVision.

Details

Applies to EthoVision XT users running the three-chamber sociability test (also known as the Crawley three-chamber test) with rodents. Incorrect cage placement or protocol sequencing can introduce location preference confounds and invalidate results.

Solution

**Restraint Cage Placement:**
1. The restraint cage (wire cup/cylinder) is placed in the two outer chambers only—never in the middle chamber.
2. Place one cage in each outer chamber, positioned symmetrically against the distal wall (the wall farthest from the divider opening).
3. Both cages must be identical in appearance and size to avoid introducing novelty or location preference bias.

**Standard Experimental Procedure:**
1. **Habituation phase:** Place the subject mouse in the middle chamber with all dividers open and allow free exploration of the entire apparatus (typically 5–10 minutes) with empty restraint cages in both outer chambers.
2. **Sociability phase:** Place a stimulus mouse (stranger 1) under one restraint cage and an inanimate object (or empty cage) under the other. Record the subject's exploration. Sociability is measured as significantly more time spent in the chamber containing the stranger mouse vs. the object.
3. **Social novelty phase:** Replace the inanimate object with a second novel mouse (stranger 2). The original stranger 1 mouse remains under the other cage. Social novelty preference is measured as significantly more time spent with stranger 2 vs. stranger 1.
4. In EthoVision, define three arena zones corresponding to the three chambers, plus optional small zones around each restraint cage for proximity/contact scoring.
5. Use zone dwell time and nose-point-to-zone proximity measures in the Analysis Profile to quantify sociability and novelty preference scores.