Cued and Contextual Fear Conditioning in Mice

Jeanne M. Wehner1, Richard A. Radcliffe2

1 University of Colorado, Boulder, Colorado, 2 University of Colorado Health Sciences Center, Denver, Colorado
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 8.5C
DOI:  10.1002/0471142301.ns0805cs27
Online Posting Date:  September, 2004
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Contextual and cued fear conditioning is a robust form of learning in which an association is made between stimuli and their aversive consequences. Fear conditioning has been used in laboratory rodents in part because it is a highly conserved form of behavior that is exhibited in both laboratory situations and in normal environments. Training requires only a single trial and this makes it adaptable to genetic, pharmacological, and biochemical studies. Clinically, it is has relevance to human behavior in that fear conditioning can be produced in humans, and damage to the amygdala prevents fear conditioning.

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Table of Contents

  • Basic Protocol 1: One‐Trial Cued and Contextual Fear Conditioning In Mice
  • Alternate Protocol 1: Pre‐Exposure Experiments
  • Alternate Protocol 2: Discrimination Protocol
  • Alternate Protocol 3: Contextual Learning without the Auditory Cue
  • Alternate Protocol 4: Immediate Shock Control Protocol
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: One‐Trial Cued and Contextual Fear Conditioning In Mice

  • 70% ethanol or 4% acetic acid solution for cleaning chambers
  • Mice (e.g., C57BL/6J, male or female)
  • Fear conditioning chambers (made in‐house from components described below). Pictures of typical fear conditioning chambers for mice (Paylor et al., ; Wehner et al., ) or young rats (Rudy and Morledge, ) are shown in Figures and . The essential components of a fear‐conditioning chamber are:
    • Shock generator and scrambler for administering shocks at various intensities
    • (0.1 to 1.0 mA) (e.g., Med Associates, Lafayette Instruments, Columbus
    • Instruments, or Coulbourn Instruments)
    • Sound generator that either delivers broad‐band clicker sounds or
    • low‐frequency tones (e.g., Med Associates, Lafayette Instruments, Columbus
    • Instruments, or Coulbourn Instruments)
  • Computer that will run Med PC software, and interface with the chambers that allow a timed program (e.g., Med Associates PC for Windows)
  • Sound‐attenuating chambers (Fig. ) equipped with a light (24 V, d.c.), a small fan that provides air circulation and white noise, and a speaker attached to the sound generator. The door of the chamber must allow viewing of animals through a window. Pairs of chambers can be easily placed next to each other (but must be visually isolated) so two subjects can be trained and tested simultaneously.
  • Transparent acrylic contextual conditioning chambers with removable grid floors and a screen top (Fig. A). The grids should be close enough together to prevent animals from slipping through the grid. For mice or young rats, the chamber dimensions are typically 26 × 21 × 10 cm. The grid floors are constructed of stainless steel rods 1.5 mm in diameter and spaced 0.5 cm from center‐to‐center for mice (Owen et al., , b; Wehner et al., ) and 1.2 cm center‐to‐center for young rats (Rudy and Morledge, ). For adult rats, this is typically a chamber that is 23.5 × 29 × 19.5 cm and the grid floor is composed of 16 stainless steel rods (2.5 mm in diameter) spaced 1.25 cm from center to center (Fanselow, ). For altered context testing, the smooth plastic floor (Fig. B) is the size of the grid floor and a rectangular plastic wall should be placed on the diagonal in the chamber.
  • Voltmeter and sound meter to check stimulus intensities
  • Scoring sheets with subject number, date, tester identity, and any other relevant information such as genotype or protocol number. Construct columns for each subject that will allow scoring of freezing behavior in 10‐sec blocks (Fig. ).
NOTE: The equipment listed above is the minimum requirement for observer‐based scoring. Automated scoring systems have been devised that rely on various methods such as photo beam interruptions (Bolivar et al., ), pressure transducers (Fitch et al., ), digital tracking systems (Kim et al., ), image subtraction methods (Anagnostaras et al., ; Marchand et al., ), a head video‐tracking system (Moita et al., ) and a proprietary motion detection system (Miller et al., ). These methods require additional equipment, some of which is commercially available and ready to use out of the box (Lafayette Instruments, Med Associates, Actimetrics, Viewpoint Life Sciences).
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  •   FigureFigure 8.5.1 Typical equipment used for contextual and cued fear conditioning. (A) A sound‐attenuating chamber holds the conditioning chamber; the exterior view is shown. (B) The inside of the sound‐attenuating chamber has a fan, light, and speaker.
  •   FigureFigure 8.5.2 Typical chambers for the context and altered contexts. (A) The contextual conditioning chamber is composed of a wire shock grid and a plastic top; the same exact chamber must be used for conditioning to the context and then scoring of freezing to the context. (B) The chamber for measurement of freezing to the altered context and the auditory cue (CS) is shown. In order to show the details for this picture, it has been removed from the outer sound‐attenuating chamber. However, for scoring in the altered context this chamber is kept inside the sound‐attenuating chamber.
  •   FigureFigure 8.5.3 Sample scoring sheet for recording freezing on testing day. Scores for several C57BL/6J and DBA/2J mice are shown. Mice are scored as “0” when they are moving and “1” if they are freezing. The number of freezes recorded is used for calculations described at the end of the .


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