Delay Fear Conditioning Protocol

General Overview
Delay conditioning is a type of associative learning task in which mice are presented with a neutral CS that is paired with an aversive US. The mice learn that the CS predicts the US and will elicited specific behavioral responses such as freezing when the CS is presented alone. Additionally, mice also learn to associate the environment in which the CS-US pairings take place with the US, and will elicit specific behavioral responses when in the environment in the absence of the CS.

Apparatus
Training and testing chambers consist of either a square or hexagon- shaped chamber. The floor of the training chamber is a gray metal grid through which the US is delivered, and the floor of the testing chamber is solid black wood. All chambers are mounted within specially designed sound attenuating shells. Half of the square chambers and half of the octagonal chambers were scented with 3 drops of vanilla extract added to the drop pan. Each chamber is lighted by a red ambient light and has speakers mounted on the wall and includes an exhaust fan and camera.

Training parameters
     Tone
          Duration: 20 sec
          Level: 70 dB, 2 kHz
     Shock
          Duration: 2 sec
          Intensity: 0.5 mA

Procedure

Habituation/Pre-exposure
On day 1 bring mice into experimental room and place individually into a training chamber. Allow paired group of mice to acclimate for 12 minutes. For the unpaired group of mice allow to acclimate for 2 minutes (120 sec); at the end of the 2 minutes present the CS (tone) for 20 sec followed by an 80 sec interval. (repeat 6 times).

Training
On day 2 bring mice into experimental room and place individually into a training chamber for 4 minutes. For the paired group, following acclimation period, begin trials: 20 second interval, followed by a 20 second tone that co-terminates with a 2 second foot shock, followed by a 60 second interval. Repeat 6 times. Following the 60 sec at the end of the last trial remove subject from chamber and return to the home cage. For the Unpaired group, following acclimation, begin trials: 38 second interval, followed by a 2 second footshock, followed by a 60 second interval. Repeat 6 times. Following the 60 sec at the end of the last trial remove mouse from chamber and return to the home cage.

Testing
On day 3 bring subject into experimental room and place individually into training chamber for 3 min. Return mouse to home cage for an ITI of 2 min. Then place mouse in test chamber (novel environment) for 3 minutes. Following acclimation begin trials: 20 second interval, followed by a 20 second tone, followed by a 60 second interval. Repeat 4 times. Following the 60 sec at the end of the last trial remove mouse from chamber and return to the home cage.

Schematic of delay fear procedure. Note that times indicated by dashed lines are not to be repeated.