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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




HMDPpheno4_Protocol

Project protocol - Contents

Workflow and sampling

 

Day
Procedure performed
Equipment
*Data collected
1
 Fear conditioning, training
fear conditioning chamber
thigmotaxis, velocity, immobility, activity burst
2
 Contextual fear test
fear conditioning chamber
thigmotaxis, velocity, immobility
3
 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



References

    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