Trace Fear Conditioning in Mice

The overall goal of the following experiment is to evaluate hippocampal dependent learning and memory in mice through the use of trace sphere conditioning. This is achieved by first training the animal to associate an auditory tone with an aversive stimulus. The next day, the animal is placed in a novel context and freezing behavior is recorded in reaction to the tone.

On the third day, the animal is returned to the original context and freezing behavior is recorded. Results show that mice trained with the shock tone pairing display more freezing behavior than those not receiving the shock. This method can be used to provide insights into hippocampal dependent learning and memory and mice, and is especially useful when used in the study of transgenic and knockout animals.

The following procedure is performed in a fear conditioning chamber housed inside a larger sound attenuating chamber. With the door to the chamber closed position the lights to reach the desired level of illumination if correctly placed the light level indicator light should be off. Use a sound meter to confirm a consistent level of background noise inside each chamber.

Next, calibrate the tone used during training. Use an external calibration device to calibrate the shock level. Place one lead on a bar and another three or four bars over.

Use the shock generator to administer the shock and calibrate until the correct level is achieved. Repeat this step for each fear conditioning chamber. Once the apparatus is prepared, transfer the subjects to a holding room separate from the testing room to reduce excessive handling.

Before testing, label the tails at least 30 minutes before testing or on the day before. When ready to begin, transfer the test mouse into a clean labeled cage for transport to the fear conditioning chamber. Place the mouse in the chamber, close the door and start the software program.

On the first day of training, mice are allowed to explore the chamber for three minutes. The software then presents a 22nd tone, followed by a 22nd trace interval that terminates in a mild foot shock. Observe the subject to confirm that they received the aversive stimuli.

A 202nd inter trial interval separates five conditioning trials. After the trials, allow the animal to remain in the testing chamber for one minute before removing, place the animal back in the transfer cage and return to the holding room. If there are additional mice to be tested in their home cage individually, house the mouse until all mice have completed testing.

Clean the test chamber with 30%isopropanol in preparation for the next animal. Repeat these steps until all mice have been trained. Return all mice to their colony room.

After the last mouse in the cohort has been tested the following day, animals are tested. For trace fear memory. On this day, replace the bedding of the transfer cages with shredded paper to change their context.

The context of the chamber is also changed. Place a foam sheet under a clear acrylic insert on the floor to alter its texture and appearance. Place an acrylic divider inside the testing area.

Change the odor by placing vanilla extract in a whey boat under the floor. Insert on this day, clean the chamber with 70%ethanol instead of 30%isopropanol between each mouse. When ready, place the mouse in the test chamber and start the program.

The session begins with a two minute baseline period followed by three 22nd tone presentations. There is a 222nd inter trial interval between each tone presentation. After the session is over, return the mice to their home cage and continue until all animals have been tested in preparation for testing on the next day.

Restore the same context as used in day one. Clean the chamber with 30%isopropanol. Contextual conditioning is conducted on day three.

Using the same transfer cages as day one, retrieve the test mouse from the holding room. Place the mouse in the testing chamber and then start the program for testing contextual memory. Run a program to record freezing behavior for eight minutes.

After this time, return the mouse to their home cage and continue testing as shown earlier, representative results show an increase in freezing behavior in animals receiving the shock tone pairing over the course of the training session. In a new context, mice that received the aversive shock stimulus had more freezing compared to the mice that did not receive the shock during the baseline tone trace and inter trial interval. When returned to the original context, animals receiving the aversive shock stimulus showed consistently higher freezing behavior over the eight minute period.

After watching this video, you should have a good understanding of how to use trace fear conditioning in order to understand hippocampal dependent learning and memory and mice.