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

Abstract

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.

Figure 1

Left: Reconstructions of the largest (black) and smallest (grey) lesions that were included in the data analysis for Experiment 5. Figures from Franklin and Paxinos (2008) used with permission. Right: Photomicrograph of a representative lesion.

Figure 2

Results from our initial conditioning protocols (Experiment 1). As expected, all animals show evidence of contextual fear conditioning. However, only delay conditioning produces specific evidence of freezing above the levels of unpaired controls (* = significantly different p<.05).

Figure 3

Results from our intermediate conditioning protocols (Experiment 2). As expected, all animals show evidence of contextual fear conditioning. However, no conditioning protocol produces specific evidence of freezing above the levels of unpaired controls.

Figure 4

Results from our final conditioning protocols (Experiment 3). As expected, all animals show evidence of contextual fear conditioning. In addition, both trace and delay conditioning produce specific evidence of freezing above the levels of unpaired controls (* = significantly different p<.05).

Figure 5

Results from hippocampus lesions on our final conditioning protocols (Experiment 4). Hippocampus lesions disrupted freezing to the auditory cue following trace conditioning, but not delay conditioning (*=p<.05) confirming that this protocol produces hippocampus-dependent trace fear conditioning in mice. In addition, freezing to the novel context, but not the conditioning context, was significantly disrupted by the lesions.