SOP of fear conditioning test



Purpose
The contextual and cued fear conditioning test assesses the ability of mice to learn and remember. In this test, a mouse is placed into a chamber and is given a pair of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric foot shock). After a delay time, the mouse is exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the exposure to conditioning chamber and cue is measured as an index of fear memory



Experimental Design
Minimum number of animals: 7 per experimental group
Age at test: 13 weeks.
Sex: males



Parameters measured
Immobility time (freezing) is measured by using video tracking system. Percentage of freezing time in 30 seconds is calculated and used as an indicator of learning/memory performance. Averaged freezing ratios during contextual testing and exposure to tone are also used.


Procedure
1. Conditioning (day1)
Each mouse is placed in a shocking chamber (white wall (box A)) for 120 s as habituation. After the habituation, 4 tone-shock pairs are given at 90 seconds intervals. The tone-shock pair consists of tone (70db, 10kHz) for 30 seconds and a foot shock of 0.5 s at 0.5 mA.






2. Context testing (day2)
Approximately 24 hours after conditioning, each mouse is placed in the box A for 6 min.

3. Cued testing (day 3)
Approximately 24 hours after contextual testing, each mouse is placed in a white transparent chamber (box B which is different from box A) for 120s in order to measure baseline data. After the measurement of baseline data, tones same as conditioned stimulus are given.

Notes
Please ensure that the mice are not handled prior to the test, except handling for cage change.


Data QC
1. If wild type mice show low performance in the learning test, look back their behavior during conditioning.
2. If mutants exhibit increased or decreased freezing time relative to wild type, look back their behavior during conditioning.


Equipment
Purchased from O'Hara & Co. Ltd, Tokyo, Japan.


Box A, chamber for conditioning
and contextual test
  Box B, chamber for cued test