Cued and Contextual Fear Conditioning for Rodents

Peter Curzon; Nathan R. Rustay; Kaitlin E. Browman

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Buccafusco JJ, editor. Methods of Behavior Analysis in Neuroscience. 2nd edition. Boca Raton (FL): CRC Press/Taylor & Francis; 2009.

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Methods of Behavior Analysis in Neuroscience. 2nd edition.

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Chapter 2Cued and Contextual Fear Conditioning for Rodents

Peter Curzon, Nathan R. Rustay, and Kaitlin E. Browman.

2.1. INTRODUCTION

Understanding what an animal learns when exposed to novelty is of great interest to behavioral neuroscientists, but it can be challenging to understand what information is acquired in a particular learning session. The behavior of an animal has to be quantified using either visual or mechanical measures of a particular response. One way of elucidating mechanisms involved in discrete learning sessions is to study associative learning processes. Simplistically, associative learning is an adaptive process that allows an organism to learn to anticipate events.

One form of associative learning that has been used in multiple species, including humans, is eye-blink conditioning. The most common species used, the rabbit, has yielded interesting results, especially in identifying and elucidating the involvement of the cerebral cortex. Similar procedures have been used in cats, rats, and humans. Another form of associative learning that has gained popularity with behavioral pharmacologists is fear conditioning. While the eye-blink procedure has overlap with context/cue fear conditioning and in many cases yields similar results, there are some basic differences between fear conditioning and eye-blink conditioning. One main difference is that eye-blink conditioning takes many more training trials to establish. Fear conditioning has gained popularity, in large part as a result of the need to characterize mutant mice and the effects of genetic alterations; therefore, this chapter primarily focuses on fear conditioning.

Fear conditioning to either a cue or a context represents a form of associative learning that has been well used in many species [1]. The majority of the experiments reported in the literature involve the mouse; however, there is also a generous proportion of the literature devoted to the rat. There are also several reports in higher species that are not covered in this chapter. In general any of the procedures described in this chapter can be used for either the rat or the mouse.

The dependent measure used in contextual and cued (delay or trace) fear conditioning is a freezing response that takes place following pairing of an unconditioned stimulus (US), such as foot shock or air puff, with a conditioned stimulus (CS), a particular context and/or such a cue. In the case of rats and mice, this US is generally a foot shock. Obviously, if in a conditioning context one administers a foot shock that is paired with a tone, there will be learning not only to the tone, but also to the context. Two types of conditioning that are typically employed are delay or trace conditioning. Delay conditioning refers to a situation in which the US is administered to co-terminate with or occur immediately after the CS. Trace conditioning differs from delay conditioning in that the US follows an empty (“trace”) interval that separates the cessation of the CS from the onset of the US. Trace conditioning adds additional complexity to delay conditioning, as the time interval between the CS and US requires the formation of a temporal relationship between the two stimuli.

In this chapter we discuss the various challenges inherent in this type of procedure in order to enable the experimenter to set the conditions to best answer the questions being posed. One of the biggest advantages of cued and contextual fear conditioning in the rodent is that they are forms of passive learning that can be used in many strains of mice and rats, even when more pronounced motor deficits are problematic in other learning assays. As a consequence of these procedural advantages, contextual fear conditioning is gaining popularity, especially in the phenotyping of transgenic mice.

2.2. CONTEXTUAL/CUED FEAR CONDITIONING: OVERVIEW

In this section we review aspects of the different conditioning assays that are crucial in conducting these tests.

2.2.1. Contextual Fear Conditioning

Contextual fear conditioning is the most basic of the conditioning procedures. It involves taking an animal and placing it in a novel environment, providing an aversive stimulus, and then removing it. When the animal is returned to the same environment, it generally will demonstrate a freezing response if it remembers and associates that environment with the aversive stimulus. Freezing is a species-specific response to fear, which has been defined as “absence of movement except for respiration.” This may last for seconds to minutes depending on the strength of the aversive stimulus, the number of presentations, and the degree of learning achieved by the subject.

2.2.2. Cued Fear Conditioning

Cued fear conditioning is similar to contextual conditioning, with one notable exception: a CS is added to the context. In order to separate context from cue conditioning some investigators provide their subjects with a preexposure trial to the context without a US. This then allows the animal to take in all the information about the context without the presence of the cue. On a second exposure to the context, the CS is presented and the animal is better able to learn the CS association because the context is not as accurate a predictor of shock as the CS (since the animal has previously experienced the context in the absence of shock). However, preexposure to the context alone is not sufficient to fully separate cue- and context-specific freezing behavior.

2.2.3. Delay and Trace Conditioning

Although delay and trace conditioning differ procedurally in the presence (trace) or absence (delay) of a time interval between the termination of the CS and US, trace conditioning uses additional brain regions in order to establish the response. Depending on the particular region of interest (or learning realm), researchers should decide on the appropriate testing paradigm. The trace interval used can range from a relatively short (2–5 sec) period, when only small learning differences in associative learning between trace and delay conditioning is observed, to quite long (45–60 sec) periods, when the association to the cue is very weak. Repeated training trials are needed for trace conditioning in order for the association between the CS and US to be formed. However, contextual learning remains strong.

2.3. BRAIN AREAS INVOLVED

The major brain areas shown to be involved in contextual and cued fear conditioning include the amygdala, hippocampus, frontal cortex, and cingulate cortex.

2.3.1. Amygdala

Attempts to identify the contribution of individual amygdaloid nuclei demonstrate that lesions to the lateral nucleus and central nucleus attenuated freezing to both contextual and auditory conditional stimuli, while lesions of the basal nuclei produced deficits in contextual and auditory fear conditioning when the damage included anterior lesions of the amygdala [2].

Evidence suggests that the basolateral amygdala complex is a critical site for fear conditioning. This observation stems, in part, from evidence demonstrating that rodents with lesions to this neuroanatomical region demonstrate a lack of freezing in the presence of cues previously paired with foot shock. An important caveat is that some studies have suggested that an intact basolateral amygdala is not essential for the formation and expression of long-term cognitive/explicit memory of contextual fear conditioning [3], but may play more of an exclusive role in cue fear conditioning.

2.3.2. Hippocampus

While learning of the context requires input from the hippocampus, especially dorsal hippocampus and CA3, experiments have shown that this input is not necessary specifically for the learning of cue associations. It has been demonstrated, however, that for trace conditioning, the hippocampus is required for learning the tone–shock association. Manipulating the interval or “gap” between the US and CS is one way studies has isolated hippocampal involvement. As the trace interval is increased from very short intervals of 1–2 sec to 15, 30, or 45 sec, the degree of associative cue learning is reduced. Also, human subjects with damage in the hippocampus have been shown to be able to acquire delay conditioning but are not able to acquire trace conditioning [4].

2.3.3. Frontal/Ventromedial/Cingulate Cortex

The frontal/cingulate cortexes are areas of attentional learning and have been shown to be involved in the acquisition of new memories [5]. Consistent with this role, lesions or pharmacological inactivation produce deficits in contextual conditioning.

Combined results indicate that there may be significant redundancy in the neuroanatomical regions mediating fear conditioning. The ability to dissect aspects of memory is one of the advantages of this type of learning paradigm. To understand some of the literature investigating the neuroanatomical underpinnings of contextual conditioning, we suggest additional reading [5–9].

2.4. BEFORE GETTING STARTED

It is important to establish the elements of the test to which the animal may respond. If, in a given environment you deliver a shock (US) to the feet of a rat or mouse at the termination of an audible sound cue (CS), the animal will learn that the testing environment (context) is unpleasant. The animal will also learn that when the auditory cue is presented there will be a shock in the near future. If this pairing is repeated, the animal’s learning generally will be stronger, and when the animal is returned to the conditioning context, will not only freeze to the context but also to the audible cue. Freezing to this auditory cue will not be specific to the conditioning environment, and can also be observed in response to the cue in a totally new environment. A challenge is dissociating how much of the learning is to the cue and how much is to the context. One way would be a subtraction process where some animals are not subjected to the audible cue, but as you will see later in this chapter, in some cases the cue may interfere with contextual learning. Therefore, separation of context and cue is ideal to establish and understand what the animal is learning [10].

2.4.1. Types of Paradigms

2.4.1.1. Contextual/Cued Fear Conditioning

It is important to understand the different methodologies and their implications before selecting the method that is best for the given research needs (see Figure 2.1 for a schematic).

FIGURE 2.1

Four basic conditioning paradigms illustrating the timing of US (aversive stimulus) presentation.

2.4.1.2. Contextual Conditioning

This occurs when an animal is placed in a new environment (chamber, cage, etc.) and is presented with a US.

2.4.1.3. Delay/Cue Fear Conditioning

This conditioning takes place when the aversive stimulus is presented at the end of a cue (CS) (light, tone, odor), and is thus paired with the aversive stimulus (US).

2.4.1.4. Trace Fear Conditioning

Trace fear conditioning is similar to delay fear conditioning except the cue is presented for a period of time and terminated. Then following a short interval (100 msec to 60 sec) an aversive stimulus is presented (Figure 2.1).

2.4.1.5. Backward Trace Conditioning

Backward trace conditioning is used as a control group to ascertain that the measure of freezing in a trace-conditioning paradigm reflects learning of the association and not some arbitrary freezing behavior. In this paradigm the CS is presented after the US has already been presented for the trial. If the animal freezes to the tone when it has been trained in the backward trace conditioning, it suggests that the freezing is due to nonassociative factors, because the tone does not predict shock.

2.5. SAMPLE EXPERIMENTS

In the following section we detail two different procedures with variations that readers may consider for conducting experiments in their laboratories.

General Considerations

Standard housing conditions are usually acceptable for animals used in conditioning. The one caveat is that the mice should be calm and healthy before testing. In the case of some strains, fighting is quite common in male mice. This is especially prevalent in C57BL/6 and in some transgenic mice strains such as the Tg2576. Many experimenters have opted to avoid males to overcome this problem, although there are some differences in responses between male and female mice. Generally, at least 8–12 animals per treatment group are needed to generate statistical significance.

Note: Always give the mice ample habituation (60–90 min) to a novel environment if you need to change the location of animals before testing.

2.5.1. Delay Cued and Contextual Fear Conditioning

2-Trial Delay Cued and Contextual Fear Conditioning

This is a standard procedure shown to produce good cue learning and contextual learning.

Place the equipment in a quiet room. It is convenient to have an anteroom in which to house the mice; however, this anteroom should be sound insulated from the testing room so that the mice in their home cages are not exposed to any auditory cues either before or after testing. If you are running more than one chamber in the room at a time, unless the separate conditioning chambers are totally isolated, it is important to have the stimuli synchronized so that any noise leakage and response to shock will not interfere across animals.

2.5.1.1. Day 1

Set up the computer control programming of the equipment for conditioning so that a house light illuminates the chamber continuously during testing.

Program a 120-sec habituation period before the first of two identical trials begins. This allows the animal to explore briefly and to take in the aspects of the chamber. A tone (auditory) cue is then presented, generally at a level of 70–80 dB (we use 80 dB) for 15–30 sec. A mild foot shock is administered during the last 2 sec of the tone presentation and co-terminates with the tone. The foot shock is generally 0.6 mA, (0.17–0.8 mA) for 1–2 sec. (The level you select will depend on your shock source; an initial shock titration experiment may be advisable.) After the shock presentation, an intertrial interval (60–210 sec) precedes a second identical trial. Following the final shock presentation, the house light should remain on for an additional 60 sec, to enable removing the mouse in a 30–60 sec time period after the last trial.

In setting up for the experiment it is preferable to run a set of mice from a single home cage all at once. This prevents previously tested mice from affecting the behavior of cage mates. Therefore, as we have four training boxes in the conditioning room, we house our mice four per cage when possible. If desirable, mice can be weighed and injected in the anteroom before bringing them into the room for the conditioning session.

Before starting, wipe out the chamber with the same solution you are using to clean the apparatus between animals to allow the first set of mice to experience the same odors as the groups that follow. We use 70% isopropyl alcohol.

The first mouse is removed from the home cage and gently placed into the conditioning chamber (repeat for the other mice in the cage). Start the training session for all the boxes in the room. The animals can be observed live or recorded. If video recording is not available, the freezing can be scored for any or all periods during training. Generally the mice will freeze when the tone comes on at the start of the second trial since they have already received one tone-shock pairing. These data can be used to assess rate of acquisition and/or effect of drug treatment in the conditioning session.
At the end of the training, remove the mice. Keep in mind that the mice have had a stressful experience and are likely to be more difficult to handle. Use caution and handle them gently to avoid influencing the consolidation process. Place the mice back in the home cage.
Between animals each cage is again cleaned/wiped out with the 70% alcohol solution and is readied for the next animal.
Try to disrupt animals as little as possible when moving them in and out of the room.

2.5.1.2. Day 2

It is important to conduct the contextual testing as similarly to the training session as possible to maximize context conditioning. This includes odor, lighting, time of day, etc. This also maximizes differences of the novel environment where changes are made to distinguish the environments. Subjects should be well habituated to a holding area before testing if they are moved. If they are housed in an anteroom, this saves time.
If the context testing is performed first, it is usual to do this at the same time of day as the training, using the same habituation procedure.
Clean the chamber as before, then place the first mouse in the chamber with the house light illuminated. For contextual conditioning testing, simply place the mouse into the illuminated training chamber for 3–5 min; there are no tone cues presented. The mouse can be observed for the presence or absence of freezing response live or recorded for later analysis. The mice should be removed promptly at the end of contextual testing and returned to the original home cage.
The testing chamber should be cleaned out as on the conditioning day.
Allow approximately 30 min before transferring them to a new location for cue testing.
If cue testing is being carried out in the same “altered conditioning chamber,” it is very important to clean out the chamber thoroughly, and it is best to use a novel odor in the chamber for subsequent cue testing
Another alternative is to transfer the mice to a novel test room and again allow 60 min for habituation. The cue testing chambers should be distinct in size, lighting intensity, background, floor texture, and odor (we use diluted vanilla extract food flavoring wiped on the floor).
The mouse is placed in the chamber and allowed to habituate for 3 min. The same intensity tone cue used in the conditioning session is then activated for the next 3 min. One additional minute of recording without the cue is taken before the animal is removed. Again the mouse freezing behavior can either be captured live or recorded for later analysis. Using a Kinder Scientific Motor Monitor, activity beam breaks are recorded and measures of freezing are derived from a computer analysis. In our studies we have used a criterion of fewer than three beam breaks in 3 or 5 sec as the criterion for freezing. In addition, simply using beam breaks as an activity score can also be useful.

2.5.2. Trace Cued and Contextual Fear Conditioning

Trace conditioning is carried out in a similar manner to delay conditioning. Differences in the procedures are related to the setup of the programming to run the conditioning phase. Generally, trace conditioning requires more trials for the animals to associate the cue with the US. Thus, for our trace-conditioning program we settled on a five trial procedure often seen in the literature.

Computer Control

The conditioning session should start with a 60–120 sec habituation period, followed by presentation of a tone cue for 30 sec. Then there is a gap (trace interval) between the end of the CS and the start of the shock (US). This trace interval can range from 2–60 sec. (Note: As the trace interval increases, the association becomes more difficult to learn. A 15-sec trace interval with five repeating trials appears to differentiate between delay conditioning and allows for reasonable associative learning.) Similar shock levels (0.5–0.75 mA) and duration (1–2 sec) are used as in delay conditioning. Following each shock, a variable intertrial interval of 90–210 sec occurs when only the house light is illuminated. A variable intertrial interval is used between trials to minimize the possibility of the animals “expecting” a new trial. Again, wait 30–60 sec after the final shock to remove the animals from the chambers. Day 2 testing for freezing to the context and cue are carried out as in delay conditioning.

2.5.3. Contextual Fear Conditioning

In cases where the experimenter is only interested in observing the fear response to the training context, the two-trial cued and contextual fear parameters can be used. In this case the tone (CS) is not presented in training. It would seem appropriate that the cue testing after the exposure to the original context would be superfluous; however, if the animals are then exposed to a novel context and freezing is measured, this measure of nonassociative freezing can be factored in the contextual freezing measure by simple subtraction.

Other Variations

The use of a variety of trial groups in which the CS and US are not paired within an experiment (e.g., in backward trace or backward conditioning) would show whether the learning taking place is a true measure of either delay or trace conditioning. These groups are appropriate behavioral controls.

2.6. DATA ANALYSIS

The data analysis is fairly simple for the context conditioning portion. Animals watched live or post-test can simply be timed for freezing individually and assessed a time of freezing. For a “normal” animal, this would lie in the 60%–80% range, however this will vary depending on the training paradigm and strain. As mentioned previously, if several animals are viewed simultaneously, one way to score freezing is to view each animal for time epochs and note whether freezing occurs or not.

2.7. SAMPLE DATA

Our initial experiments using delay and trace conditioning revealed some of the considerations highlighted in the previous sections. We will present some of those data and discuss some of these points. One would expect only mice that have received shock in the initial training using a tone CS to show freezing to the original context. Then, when in a novel environment for the cue test all the mice would show initial increased activity, and when presented with the CS again only mice that have made an association would demonstrate freezing. Also, the magnitude of the freezing response would demonstrate the extent of that “learned association.”

To demonstrate that mice learn this association we ran three groups of C57BL/6 mice using a five trial trace-conditioning paradigm with a 30-sec trace interval. The groups included a control group that did not receive shock during training and two groups of mice receiving shock, one at 0.17 mA and one at 0.35 mA. All mice were then tested 24 hr later in the conditioning chamber for contextual learning, followed by placement in a novel chamber to assess cue learning. As can be seen in Figure 2.2, the mice that did not receive shock during training did not freeze when assessed in the 5 min context testing session in the conditioning chamber 24 hr following training, whereas the animals that received shock froze in a significant shock related fashion. When later placed in the novel environment all mice showed some freezing to the novel environment in the first 3 min before the cue was presented, with the 0.35 mA shock group showing slightly more freezing. Following presentation of the original tone (CS) for the next 3 min there was a small increase in freezing that was not significant in the no-shock group (nonassociative freezing, which is discussed later), but there was a significant increase in the groups receiving shock, which indicates learning of the tone–shock association.

FIGURE 2.2

(A) A measure of contextual learning, showing the magnitude of context freezing times in the original training context 24 hr post training without shock (ns), and that freezing times increase in relation to the shock level. (B) A measure of cue learning, (more...)

Also studied was a direct comparison between C57BL/6 mice from two different suppliers, Jackson Labs and Harlan Sprague Dawley, Inc., keeping all environmental conditions equal. The mice were trained with five trials consisting of a 30 sec CS tone of 80 dB, and a shock (0.35 mA) administered for 2 sec, in either a delay conditioning procedure (US in the last 2 sec of the CS) or in a 30-sec trace conditioning procedure (US 30 sec following the termination of the CS).

As can be seen in Figure 2.3, the Harlan mice exhibited more freezing than the Jax (Jackson Labs) mice in the original context; however, the Harlan and Jax mice were similar when trained with five trials of trace conditioning.

FIGURE 2.3

Depicts the difference in freezing times (mean, +/− SEM) between C57BL/6 mice obtained from Jackson Labs (Jax) and Harlan Sprague Dawley (Harlan) when trained and tested for contextual conditioning 24 hr later.

These mice were also tested for cue conditioning in the Kinder Scientific Motor Monitors. In order to clearly show some supplier differences, Figure 2.4 shows the activity scores as beam breaks for the cue testing in a novel environment. It can be seen in the delay conditioning paradigm that mice from both Harlan and Jax demonstrate less activity when the tone is presented. However, the Jax mice recover their activity levels once the tone is turned off during the last minute of the session (see arrow), showing good associative learning; the Harlan mice do not show this. In addition, trace conditioned mice do not demonstrate this recovery of activity when the cue is turned off (minute 7). This could be interpreted as the demonstration that the mice trained in the trace conditioning paradigm learned that “tone off” is as good of a predictor of shock as is “tone on.” Also, Jax mice show more activity than the Harlan mice in the first 3 min of the session.

FIGURE 2.4

Illustration of differences in freezing times (mean, +/− SEM) between C57BL/6 mice obtained from Jackson Labs (Jax) and Harlan Sprague Dawley (Harlan) when trained and tested for response to the cue when presented in a novel environment 24 hr (more...)

2.8. NONASSOCIATIVE FREEZING COMPLICATIONS

It has been shown that exposing rodents to foot shock will sometimes produce generalized, or nonassociative freezing to unconditioned stimuli, such as when placed in a novel environment [11]. In our experience, we can see increased freezing to the tone stimulus in the novel environment in animals that have received contextual conditioning, i.e., those that have not been exposed to the tone during conditioning. To demonstrate these nonassociative freezing effects of the tone in our trace conditioning paradigm, we ran an experiment using groups of mice preexposed to the tone CS, thereby establishing it as a neutral stimulus. The experimental setup is seen below: the preexposure, conditioning, and context tests take place in the conditioning chamber, and the cue test takes place in a novel chamber.

Group	Preexposure	Conditioning	Context Test	Followed by Cue Test
	Day 1	Day 2	Day 3
Tone—Paired	Context Only	Tone and Shock	5 min Context	Cue Test with Tone
Tone—Unpaired	Context and Tone	Shock Only	5 min Context	Cue Test with Tone

On day 1, an acclimation day, all mice were placed into the conditioning chamber. The “tone—unpaired” group was exposed to five 30-sec CS (tone 80 dB), and another “tone—paired” group of mice was exposed to the conditioning chamber without the tone. On day 2, both groups were again placed in the conditioning chamber. The unpaired tone group received five contextual conditioning (no tone) trials. The paired tone group received five trials of trace conditioning with a trace interval of 15 sec. The US was a 2-sec shock of 0.78 mA for both groups. This design resulted in both groups receiving an equal number of exposures to the tone (CS), the shock (US), and to the amount of time in the conditioning chamber. The only difference in the treatment of the two groups was the presence (paired) or absence (unpaired) of the tone on day 2 when the shock and tone association was presented. On day 3, both groups were tested first for contextual conditioning in the conditioning context for 5 min, then in a novel chamber with 3 min of no tone, 3 min of tone, and 1 min recovery (no tone).

As can be seen in the Figure 2.5, both groups exhibited the same level of freezing in the conditioning context; however, the mice with a paired association exhibited a greater response when the CS was presented in the novel context.

FIGURE 2.5

Illustration showing that nonassociative freezing to the tone cue can be reduced by preexposure to the tone in the unpaired group. Both groups had equal exposure to the tone cue, but mice in the unpaired group received tone exposure on the day prior to (more...)

Statistical Analysis

Generally all that is needed is either a one- or two-way analysis of variance (ANOVA) with appropriate post hoc analysis comparing the various treatment groups using either the raw or percent freezing scores of the contextual freezing. In addition, this analysis may be performed on the data from the cued conditioning scores.

2.9. FINAL NOTE

We hope from reading this chapter the reader has gained a basic understanding of the cued and contextual fear conditioning paradigms used in rodents, and comprehension of the difference between trace and delay fear conditioning. This chapter is just a stepping-off point—there is a vast amount of literature that has been published on the involvement of the various brain regions responsible for associative learning, as well as the differences between trace and delay conditioning models. Using this information, one should easily be able to set up equipment to carry out studies that will lead to productive research. There is an extensive addendum to help you start testing.

2.10. ADDENDUM

2.10.1. Available Equipment Options

There are several options when it comes to choosing the equipment necessary to demonstrate a reliable cue and contextual fear response. The equipment does not have to be terribly sophisticated and a number of labs that made their own chambers are still using the custom equipment.

It is most important to be able to monitor the animals either visually (live or through the use of a video system) or by incorporating a movement monitoring system to reliably measure freezing behavior. Miniature video cameras are available, (for example, CCTVOne) and easily located inside an isolation cubicle to monitor the animal or record the session on tape or DVD. Recording of the behavior enables the researcher to review and reanalyze an experiment, which can be advantageous. Some laboratories perform live visual monitoring of animals during an experiment and when testing multiple animals simultaneously, and will time sample each chamber.

2.10.2. Conditioning Chambers

Chamber Size

The size of the chamber is not critical, but the chamber should be constructed to be easily cleaned and have a way to easily view the subject. In addition, if the same chamber is being used for both contextual and cued conditioning, it should be easily adaptable to making the context distinctly different when changing from contextual to cued conditioning. Making the chamber different should involve changes in the floor (e.g., from stainless steel bars to a solid plastic or equivalent), and changes in the inside dimensions by the addition of a diagonal divider. In addition, it is helpful to change the odor by using a diluted food essence.

Equipment Examples

In this section we list some suppliers of fear conditioning equipment (Table 2.1).

TABLE 2.1

Names and Addresses of Vendors

Kinder Scientific

Our lab uses equipment available from Kinder Scientific. The conditioning chamber we use consists of one side of the active avoidance system and is therefore used for contextual freezing testing (Figure 2.6). We run four chambers at a time that are under the control of one computer to maximize throughput. Each chamber is equipped with a video camera and the video output is fed into a splitter to record multiple animals on one DVD recorder.

FIGURE 2.6

Kinder Scientific Learning and Memory Avoidance Systems.

Coulbourn Habitest

Testing can also be achieved by using a Coulbourn Habitest chamber for conditioning and contextual fear testing. Cue testing can then be carried out in another chamber equipped with the same audible cue or by altering the conditioning chamber.

Med Associates

Med Associates will sell a complete package for contextual fear conditioning and can include an infrared video analysis system that detects freezing (Figure 2.7). This analysis has been shown to be equivalent to visual scoring in a paper by Contarino and colleagues [12]. There are confounds to learning when both cued and contextual conditioning are measured in the same chamber where the conditioning took place.

FIGURE 2.7

MedAssociates Contextual Fear System including freezing detector.

San Diego Instruments

San Diego Instruments also provides a stand-alone Freeze Monitor that uses photocells in a higher density than a normal activity chamber arrangement. They also provide a separate Freeze Monitor context enclosure to detect nonassociative freezing.

Clever Systems, Inc

Freeze Scan^® is a software system for automatically detecting freezing states in rodents and fulfills the demand for high throughput screening. Freeze Scan^® is a video-based tool that provides precise motion control for accurate freezing detection. Freeze Scan^® accepts video taken from different views in a confined chamber. It precisely detects the onset and completion of the freezing behavior of a rodent. Its output is a sequential list of the occurrences of the freezing behaviors. Further statistics can be analyzed from this output data. Freeze Scan^® has the capability to set the same intervals generated by the tone, shock, or light control program. Clever Systems, Inc. can also provide the hardware for tone, shock, and light control for use in your chamber, and thus Freeze Scan^® can synchronously work with the control program and provide accurate freezing state results based on these intervals.

2.10.3. Considerations for the US (Shockers)

The level of shock produced as the US will vary to a great degree on the shock source (manufacturer). In order to present a standard shock level to each mouse or rat it is best to use a DC constant current source. Most commercially available shockers, such as those from Coulbourn Instruments, are square wave sources. This means that in contrast to AC or sine wave sources, there is an instantaneous rise time and off time that is more aversive at a lower current level. A scrambled shock is switched between the bars of the floor; the current is applied separately to each bar in a span of 8 bars that are sequenced over a 32-bar floor. The Kinder Scientific Active Avoidance box comes with an internal square wave constant current source. In the case of other chambers, external shock sources need to be supplied. Discrepancies in shock levels used in different laboratories usually can be attributed to the type of shocker used. The best measure is to actually observe the animal and gradually increase the shock to a level where the animal first vocalizes and rapidly runs around the cage. This level is lower than that where the animals start jumping to avoid the shock, although some strains may show a hyperresponsivity to an initial shock.

Note: It is advisable to use an oscilloscope to measure and view the shock output at the level of the grid in the test apparatus. This helps to reduce the possibility of not obtaining a reproducible effect.

2.10.4. Cued Conditioning Measurement

The Kinder Scientific activity monitoring system has a board containing LED photocells that surrounds the chamber and measures beam breaks surrounding a Plexiglas chamber (7.5 in × 14 in) with a flat, white plastic tray floor that can be used for measuring conditioned association. The ceiling contains a lamp and a Mallory Sonalert^® sound source identical to the ones used in training. We enclosed the chambers in a box made of pink insulation and the chambers were adapted to allow video recording of the animal behavior. You may also consider using the Kinder Scientific cued fear conditioning chamber or any of the other conditioning setups. However, it must be emphasized that the animal not only learns about the immediate context into which it was placed when it received the aversive stimulus, but it also learns all the events and places leading up to being placed in that context. To reduce any generalization from the conditioning context to the novel context, it is preferable to carry out the cue testing in a separate room with new visual, tactile, and olfactory cues [13]. In our laboratory, the assessment of cued conditioning is conducted in a totally different sized room in the facility.

2.10.5. Measuring Freezing Behavior

Two options are available for measuring freezing behavior.

2.10.5.1. Hand Tallying Method

This method can be carried out in a few different ways. If the animals’ behavior is recorded, individual animals can be observed and continuously monitored for freezing. If it is desirable to monitor more than one animal simultaneously, then a sampling system can be adopted. For example, one method is to observe each cage sequentially for 5 sec and simply note whether freezing is occurring (yes or no) in each chamber for each 5-sec bin. These data can be recorded on a chart. If six cages were scored simultaneously for a 5-min session you would have 10 bins per animal. If an animal demonstrated freezing in five of the bins, this would translate to a 50% freezing score. Another similar approach is to monitor each animal once every 5–10 sec and note whether the animal is freezing at the exact moment of observation. Yes or no scores can be tabulated in an identical way as in the above approach. These sampling methods may not be as accurate as constant measuring, but if carried out uniformly they will yield reproducible results. In fact, it has been shown that scores obtained from these sampling methods correlate very highly with scores obtained from continuous sampling [12].

2.10.5.2. Activity Monitoring Method

This method takes into account distance traveled and/or the number of beams broken. In the Kinder analysis, the movement of the central part (centroid) of the animal is taken into account to calculate a “rest time.” An animal is considered at rest when there are fewer than a specific number of beam breaks in a specified period (the numbers can be specified by the experimenter). These time epochs are then counted up to give a rest time total in seconds. We consider an animal to be at rest or “freezing” if there are fewer than three beam breaks in 5 sec. The total freezing score is validated by independent observation and timing of the freezing response compared to the computer analysis of the rest time.

San Diego Instruments has a similar system (Freeze Monitor) that uses a concentrated number of infrared photo beams measuring beam breaks. The analysis is similar to that described above.

Infrared Video: A Fire-Wire^® system is available from Med Associates, Inc. that can be used to detect freezing in the animal. This is a computer-based system that has been validated. See Anagnostaras and colleagues [14].

Clever Systems also has a sophisticated online video analysis. This system uses a similar method of pixel analysis of the streamed video frames.

Note: Whatever system you choose, it is always necessary to run a set of animals to demonstrate that any instrumentation analysis is producing data that correlates highly with those obtained using a visual scoring method.

2.10.6. Animal Strain Considerations

Many different mouse strains have been used for cued/contextual fear conditioning. Behavioral phenotype can affect the magnitude of the freezing behavior or the level of associative learning. In some strains of mice retinal degeneration is a background genetic defect that may or may not affect contextual freezing. In other cases, hearing deficits develop as the animal ages. Some mouse strains may demonstrate reduced responsiveness to foot shock. Therefore, when designing the experiment, the strain, cue type or magnitude, and aversive conditioning levels should be considered. In many studies that have reported optimal learning of the context and cue, researchers have used the C57BL/6 mouse with a white noise, tone, or clicker cue, and 0.4–0.6 mA shock level. Comparison of not only strain but also animal supplier may also yield different results and can be explored [15].

In the case of rats (especially aged rats), quantification of the freezing response as well as variability in audition and response to shock could also affect the behavioral measures. Sprague Dawley rats tend to freeze more than other strains, and some of these problems have been overcome by using transmitters to measure changes in heart rate as the measure of conditioned fear.

2.10.7. Auditory Cue Considerations

The superficial selection of the CS for the apparatus appears to be wide ranging. Studies have been conducted with clickers, white noise, and pure tones generally between 70 and 85 dB. RadioShack makes a convenient meter that will give a good reading of dB levels. It should be set on continuous with “A” weighting to allow for mean dB levels. The noise-producing device is most effective if it can fill the chamber without noise “dead spots” to ensure that subjects will receive the CS regardless of their position in the chamber.

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