Developing and validating trace fear conditioning protocols in C57BL/6 mice

J Neurosci Methods. 2014 Jan 30:222:111-7. doi: 10.1016/j.jneumeth.2013.11.005. Epub 2013 Nov 20.


Background: Classical fear conditioning is commonly used to study the biology of fear, anxiety and memory. Previous research demonstrated that delay conditioning requires a neural circuit involving the amygdala, but not usually the hippocampus. Trace and contextual fear conditioning require the amygdala and hippocampus. While these paradigms were developed primarily using rat models, they are increasingly being used in mice.

New method: The current studies develop trace fear conditioning and control paradigms to allow for the assessment of trace and delay fear conditioning in C57BL/6N mice. Our initial protocol yielded clear delay and contextual conditioning. However, trace conditioning failed to differentiate from an unpaired group and was not hippocampus-dependent. These results suggested that the protocol needed to be modified to specifically accommodate trace conditioning the mice. In order to reduce unconditioned freezing and increase learning, the final protocol was developed by decreasing the intensity of the tone and by increasing the inter-trial interval.

Results: Our final protocol produced trace conditioned freezing that was significantly greater than that followed unpaired stimulus exposure and was disrupted by hippocampus lesions.

Comparison with existing methods: A review of the literature produced 90 articles using trace conditioning in mice. Few of those articles used any kind of behavioral control group, which is required to rule out non-associative factors causing fearful behavior. Fewer used unpaired groups involving tones and shocks within a session, which is the optimal control group.

Conclusions: Our final trace conditioning protocol can be used in future studies examining genetically modified C57BL/6N mice.

Keywords: Associative learning; Behavior; Conditioning; Fear; Mouse.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Acoustic Stimulation
  • Analysis of Variance
  • Animals
  • Conditioning, Psychological* / physiology
  • Electroshock
  • Fear* / physiology
  • Hippocampus / pathology
  • Hippocampus / physiopathology
  • Learning / physiology
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Motor Activity / physiology
  • Psychological Techniques*
  • Random Allocation
  • Time Factors