Brown Institute for Brain Science

Fear Conditioning (contextual, cued, & extinction)

Rationale of Test

Fear conditioning a commonly used paradigm to assess an animals ability to learn basic associations. This testing paradigm is commonly employed to study the functioning of the hippocampal memory system (contextual fear conditioning), basic pavlovian conditioning that relies heavily upon the amygdala (cued fear conditioning), and/or the ability to learn to suppress a fear response once it has been learned (fear extinction). These models are employed to study basic memory function as well as to model disease in which fear and emotionality are key components (PTSD, anxiety disorders, phobias, and the beneficial effects various therapeutic interventions).

Apparatus

Fear conditioning is carried out in fear conditioning chambers equipped with a shock grid floor, speaker, and digital video camera. Chambers are set up inside of sound attenuated cubicles and connected to a controller computer. Chambers are cleaned with a scented ethanol solution between mice to eliminate odors left by the previous animal. For cued conditioning, chambers of differing shape, dimensions, and with a different floor and different scented ethanol are used to maximize the difference between contexts.

Procedure

The conditioning apparatus consists of a mouse shock-chamber set up in a sound attenuated box and scented with peppermint odor (0.1% peppermint). On day 1, the conditioning day, following a 2.5 min acclimation period to the conditioning chamber, mice receive three conditioning trials consisting of a 30 second presentation of a (5 kHz, 70-dB) tone (CS) that co-terminates with a 0.7-mA foot shock (US) delivered through the grid floor during the last 1.0 sec of the tone. Each conditioning trial is separated by a 40 second inter-trial interval. Following conditioning, mice are returned to their home cages. The mouse’s behavior is digitally recorded during CS presentations for subsequent quantification. Time spent "freezing" prior to and during the presentation of the tone CS is measured during the CS presentation as well as during a 30-sec baseline period prior to the first tone trial. This latter measure serves as an assay for both unconditioned effects of the context and tone on general activity levels. Following tone testing, mice are returned to their home cages. Memory for the context and the tone are evaluated on day 2 and 3 respectively (~24 and 48 hr following conditioning). For the context test, mice are placed into the conditioning chamber and allowed to explore for 1 min, after which freezing to the context is assessed for the remaining 4.5 min. For the tone test, mice are placed in a novel chamber (circular in shape, with red walls, and scented with Lemon odor), allowed to acclimate to the chamber for 2.5 min, and then presented with 3 tones (30 sec, 5 kHz, 70 dB, ITI = 40 sec). Freezing is evaluated during the 2.5 min acclimation period, during each presentation of the tone CS, and during the 40-sec inter-trial interval. Following memory tests, animals are returned to their home cage and colony. For extinction training, mice are place in the novel cue chamber and repeatedly presented with the conditioning tone for 40 trials at 40sec inter-trial interval. Freezing behavior to the tone is assessed for each mouse, and changes in freezing are used as indicators of the mouse’s ability to suppress a learned fear response. Alternatively, mice can be placed in the context and allowed to freely explore for a period of 30 minutes. During this trial, no tones or shocks are administered and freezing behavior is assessed on a minute-by-minute basis.

Interpretation

During the memory tasks, decreased freezing behavior is used as an indicator of an animals recognition of a potentially aversive stimulus, and thus an indicator of memory function. Decreased freezing during the context portion of the task is used to assess hippocampus-dependent memory. Decreased freezing during the cued-portion of the task is used to assess amygdala-dependent memory. Rate of decrease in freezing behavior during the extinction task is used to assess efficacy of the prefrontal cortex to suppress amygdala activity. The cued portion of the task is critical for the interpretation of the context portion of the task, as alterations in freezing behavior during the context test can be significantly influences by additional impairments in basic associative learning processes.

Relevant Controls

Care should be taken to insure no differences between groups in pain sensitivity, sensory function (vision & audition), and basal activity.

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