HMDPpheno4 project protocol

Fear conditioning in males of 94 strains of mice in the Hybrid Mouse Diversity Panel (HMDP)   (2011)

Park CC, Gale GD, Lusis AJ, Smith DJ
With: Khan AH, de Jong S, Ghazalpour A, Bennett BJ, Farber CR, Langfelder P, Lin A, Eskin E, Horvath S, Ophoff RA


Project protocol - Contents

Workflow and sampling


Procedure performed
*Data collected
Fear conditioning, training
fear conditioning chamber
thigmotaxis, velocity, immobility, activity burst
Contextual fear test
fear conditioning chamber
thigmotaxis, velocity, immobility
Cued fear test
activity monitoring system
thigmotaxis, velocity, immobility

* Additional data available in HMDPpheno4_suppdata.xlxs

Equipment and supplies

  • Modified Gemini Avoidance System (San Diego Instruments, San Diego CA) [25 cm x 20 cm conditioning chamber with grid floors and white plexiglass]
  • Activity monitoring chamber [50 cm x 25 cm]
  • Video camera mounted above each test chamber
  • EthnoVision Pro tracking system and software (Noldus Information Technology)

Reagents and solutions

  • 70% ethanol for cleaning

Acclimation to test conditions

Mice were allowed 14 days to become accustomed to the housing facility. On each test day, mice were placed in a holding room for a 30-min acclimation period prior to testing where auditory background white noise (80 dB) was delivered through overhead speakers. [Mice tested between 1000h and 1600h.]

Procedure: Fear conditioning, contextual fear, and cued fear

Mice are exposed to a fear conditioning protocol followed by two independent memory tests: contextual and cued fear. Behavior is recorded digitally from a camera mounted above each test chamber then digitized at 15 frames/s. Eighty-two unique endpoints are quantified automatically with software (for more details about this procedure, equipment, and data analysis see Park et al., 2011 and Gale et al., 2009). See HMDPpheno4_suppdata.xlxs for all endpoint data.

    1. On day 1, mice are placed in the fear conditioning chamber; following a 3-min exploration period, mice receive 3 tone-shock pairings, where auditory conditional stimuli (CS) = 2000 Hz, 15 s, 80 dB with co-termination of a footshock unconditional stimulus (US) = 0.75 mA, 1 s; pairings are delivered with an inter-trial interval of 1 min; mice are removed from test chamber 2 min following final US.
    2. On day 2, contextual fear is assessed in the same fear conditioning chamber as day 1; neither CS nor US are presented during the 8-min test.
    3. On day 3, cued fear is assessed following a contextual shift (mice placed in a novel activity chamber); following a 3-min exploration period, mice receive a series of ten CS presentations (1 min inter-trial intervals); US are not presented; mice are removed from the chamber 1 min following the final CS (no US presented during this test).

Definitions and calculations

  • Conditional stimulus (CS): tone (2000 Hz, 15 s, 80 dB)
  • Unconditional stimulus (US): footshock (0.75 mA, 1s)

Data collected by investigator

  • thigmotaxis (wall preference)
  • velocity
  • *path shape (meander)
  • immobility
  • *mobility
  • *habituation

* Available in Supplementary data file only HMDPpheno4_suppdata.xlxs


    Gale GD, Yazdi RD, Khan AH, Lusis AJ, Davis RC, Smith DJ. A genome-wide panel of congenic mice reveals widespread epistasis of behavior quantitative trait loci. Mol Psychiatry. 2009 Jun;14(6):631-45. doi: 10.1038/mp.2008.4. Epub 2008 Apr 1. PubMed 18379576   FullText