Trace Fear Conditioning in Mice

Summary

In the following experiment we describe a protocol for trace fear conditioning in mice. This type of associative memory includes a trace period that separates the neutral stimulus and the unconditioned stimulus.

Abstract

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings between a neutral stimulus and an unconditioned stimulus. There is a 20 sec trace period that separates each conditioning trial. On the following day freezing is measured during presentation of the conditioned stimulus (CS) and trace period. On the third day there is an 8 min test to measure contextual memory. The representative results are from mice that were presented with the aversive unconditioned stimulus (shock) compared to mice that received the tone presentations without the unconditioned stimulus. Trace fear conditioning has been successfully used to detect subtle learning and memory deficits and enhancements in mice that are not found with other fear conditioning methods. This type of fear conditioning is believed to be dependent upon connections between the medial prefrontal cortex and the hippocampus. One current controversy is whether this method is believed to be amygdala-independent. Therefore, other fear conditioning testing is needed to examine amygdala-dependent learning and memory effects, such as through the delay fear conditioning.

Introduction

In fear conditioning a neutral stimulus (NS) is paired with an aversive unconditioned stimulus (US). The NS is normally a tone and becomes a conditioned stimulus (CS) through repeated pairings with the US. The CS can then elicit a conditioned response (CR), such as freezing, in the absence of the aversive US. A commonly used fear conditioning protocol is delay conditioning. In this protocol the onset of the NS and the US is contiguous or with some overlap in stimulus presentation. Even though delay fear conditioning is one of the most commonly used types of temporal associative conditioning, there are several other types of associative conditioning temporal arrangements: simultaneous conditioning, backward conditioning, and trace conditioning¹. In trace fear conditioning there is a stimulus-free interval between the NS and the US of several seconds resulting in a "trace" period.

Several studies have reported deficits in trace fear conditioning when neurotoxic lesions are produced in the structures that input into the hippocampus^2-5 or when pharmacological agents are used to block receptor function in the hippocampus. Lesion to the hippocampus results in deficits in trace conditioning and contextual conditioning, but does not impair delay fear conditioning⁸. There are several benefits to using trace fear conditioning. The fear conditioning protocol can be achieved over a three-day testing period and allows for hippocampal-dependent memory that is not spatially dependent. Trace fear conditioning can be used as a complementary test to the Morris water maze, novel object recognition test, or other maze tests in investigating hippocampal-dependent memory.

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Protocol

The mice used in the following experiment were generated and housed at Baylor University at an ambient temperature of 22 °C, with a 14 hr light and 10 hr dark (20:00 to 6:00 hr) diurnal cycle. The mice were given ad libitum access to food and water. All procedures to the mice were in compliance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and the animal protocol was approved by Baylor University Animal Care and Use Committee.

Overview

The trace conditioned fear task is based on procedures described by Wiltgen and colleagues⁹.

1. Preparation of Equipment

The fear conditioning apparatus chamber (26 cm x 22 cm x 18 cm) consists of two sides that are acrylic, two sides that are metal, and a grid floor bottom which is used to deliver a mild foot shock. The testing chamber is housed in a sound attenuated chamber. The chamber is also light tight to prevent outside light from influencing the motion detection software.

1. Calibrate the shock levels, light levels, and sound intensity levels for the testing chamber. Measure the background level of the testing chamber. The background noise in this chamber is 65 dB. Use a sound meter to measure this level. The meter should be set to 70 dB, set to C, and to detection setting slow.
   1. Calibrate the shock level to 0.5 mA. Use an external calibration device to correctly calibrate the shock level (see Materials Table). The internal measurement of the shock generator is not accurate. The shock generator administers a scrambled shock which cannot be accurately measured by a standard amp meter.
   2. Place one lead on one of the grid bars and place the other lead 3 or 4 bars over. Use the shock generator to administer the shock. Adjust the shock level until the correct level is achieved. Do this for each fear conditioning chamber.
   3. Close the door to the chamber when measuring the light level of the chamber. Calibrate the light level to approximately 1.0. This is a number specific to the FreezeFrame software used in this experiment. An external light meter will read this as 2 lux. The light level can be adjusted by moving the location of the house light or by adjusting the condenser of the lens. Make sure to tighten the adjusting screws for the lens after adjusting.
   4. Calibrate the sound decibel level to 85 dB. Use an external decibel meter inside the testing cage to calibrate the decibel level (see Materials Table). The sound presented will be a 2,700 Hz tone. Note: If using mice older than 6 months old it may be better to use white noise since older mice can have hearing deficits .
2. After the apparatus is ready take the subjects to a separate holding room. Note: Do not house the mouse in the same room as the testing room.
3. Label the tail of the mice that will be tested in order of testing. It is best to reduce excessive handling before testing. Alternatively, the tails can be labeled the day before the experiment to reduce handling stress. After the mice have been labeled allow them to acclimate to the room for 30 min. Have extra clean cages for housing the mice after testing is completed.

2. Trace Conditioning Day 1

1. Remove each mouse from the cage and place them in individual cages for transport to the fear conditioning chamber. Use clean bedding for each cage. Place a sticky note on the transfer cage to keep the order of mice testing correct. Note: If the mice are singly housed then they can be transported in their home cage.
2. Place the mouse in the testing chamber and close the door. Start the software program.
3. On the training day, allow the mice to explore the chamber for 3 min. The software then presents a 20 sec tone (85 dB, 2,700 Hz) to the animal. After a trace period of 20 sec a mild shock (2 sec, 0.5 mA) is administered to the animal.
4. Record the reaction of the subject to confirm that they received the aversive stimuli by viewing the video. A 200 sec inter-trial interval separates the 5 conditioning trials. Each trial consists of a 20 sec tone followed by a 20 sec delay then a shock.
5. After testing is completed allow the animal to remain in the testing chamber for 1 min before removing it from the testing cage.
6. Place the animal back in the transfer cage and return it to its home cage. If there are additional mice in their home cage then individually house the mouse until all mice complete testing. This will reduce the stress to the other mice that have not been tested. An alternative solution would be to singly house all mice one week prior to testing to reduce the influence of continuously removing mice from one cage.
7. Clean the testing chamber with 30% isopropanol after each animal is tested.
8. Repeat steps 2.2-2.7 for all mice in the testing cohort.
9. Return all mice to their colony room after the last mouse in the cohort has been tested.

3. Trace Fear Conditioning Day 2: Trace Memory Testing

1. Trace memory testing will occur on day 2. In this protocol there are 3 tone presentations. Place the mice in a new context for the trace conditioning test.
2. Prepare the software to run a program with a 2 min baseline period followed by three 20 sec tone presentations. There is s a 220 sec ITI between each tone presentation.
3. For the new context condition, place clear acrylic inserts on the floor of the chamber to alter the shape, texture, and color of the conditioning chamber.
4. Change the odor in the chamber by placing vanilla extract in a weigh boat under the floor insert.
5. Clean the chamber with 70% ethanol instead of 30% isopropanol. Note: This will help to create a novel context.
6. Bring the mice to the holding room and relabel their tails for testing if necessary.
7. Prepare novel context transfer cages by replacing the bedding with shredded paper. Note: This will help in creating a novel context.
8. Place the mice in the testing chamber then start the program. Clean the chamber with 70% ethanol after testing is completed.
9. Return the mice to their home cage after the trace conditioning is completed. Return all mice to their colony room when all mice have been tested.

4. Trace Fear Conditioning Day 3: Contextual Memory Testing

1. On the third day contextual conditioning is conducted. Prepare the software to run a program to record freezing behavior for 8 min.
2. Clean the chamber with 30% isopropanol before testing and after testing each mouse. The context should be identical to that of day 1. The transfer cages should be the same as in day 1.
3. Bring mice to the holding room and label their tails for testing.
4. Place each mouse in the testing chamber then start the program. Clean the chamber with 30% isopropanol after testing is completed.
5. Return all mice to their colony room when completed.

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Results

For the representative results we present data from control C57BL/6J adult mice that received the neutral stimulus pairings with the unconditioned stimulus (shock condition) compared to mice that received the neutral stimulus but did not receive the unconditioned stimulus (no shock condition). It is important to run this condition when first setting up this behavioral test to determine whether the protocol has been performed correctly.

The data in Figure 1 represent the traini...

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Discussion

There have been several studies that have elucidated the neural circuitry that underlies trace fear conditioning. Trace fear conditioning is believed to involve the CA1 of the hippocampus^12-14. There is also evidence that the medial prefrontal cortex (mPFC) plays a large role in trace eye-blink conditioning¹⁵, and the mPFC has been found to be involved in trace fear conditioning. One study found that mPFC neurons provide sustained activity during the trace period, thus providing a structure that can...

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Materials

Name	Company	Catalog Number	Comments
FreezeFrame	Coulbourn
30% Isopropanol			Purchase 90% isopropanol and dilute it down to 30%
70% Ethanol
Amp-meter	Med-Associates	ENV-420	Windows XP, Vista, and 7 Compatible (32-bit only)
Digital Sound Level Meter		33-2055
Vanilla Extract	McCormick Pure Vanilla Extract
Sticky Notes	Post-it		3 in x 3 in