Pavlovian fear conditioning has become an essential behavioral task in a wide array of neuroscience research avenues. Increasingly, researchers from a variety of fields of study are turning to fear conditioning tasks to probe for behavioral and cognitive phenotypes following pharmacological, genetic, and other experimental manipulations. Delay tone fear conditioning has become a widely used assay as it allows for the assessment of “hippocampus-independent” tone fear and “hippocampus-dependent” context fear in the same animal. The main advantage of this procedure is that these two different types of fear can be tested independently. However, accurate assessment of these two types of fear makes the assumption that one does not interact with and confound the measurement of the other.
In a tone fear conditioning procedure, fear is simultaneously acquired for both the tone and contextual cues. In delay tone conditioning, acquisition of tone fear has been shown to depend on synaptic plasticity of auditory inputs into the amygdala (Medina et al., 2002) Contextual fear acquisition depends on synaptic plasticity in the amygdala as well as additional neural substrates such as the hippocampus, which are thought to mediate the integration of multimodal cues into a “contextual representation” that can function as a conditional stimulus (CS) for amygdalar circuits (Kim and Fanselow, 1992; Maren and Fanselow, 1995). Context fear is assessed by returning the animal to the original training context for a “context test.” Tone fear is usually assessed by placing the animal in a distinctly different context where the tone is presented during a “tone test.” The primary behavioral measure of fear is the percentage of time an animal spends “freezing,” which is a species-specific defensive response in the rodent characterized by complete immobility. Differences in the level of freezing during tone presentations are used to infer alterations in the acquisition or expression of tone fear.
A critical unresolved issue in the measurement of tone fear is the extent to which baseline levels of freezing observed prior to tone presentation influences the measurement of tone freezing. Baseline freezing is usually driven by fear of the training context that has generalized to the testing chamber due to similarities between the two apparatuses. For this reason most researchers make the training and testing chambers as different as possible. The shift in context for the tone test attempts to isolate expression of tone fear by eliminating the expression of context fear. However, baseline fear is rarely reduced to zero, especially in mice which tend to show a higher degree of context generalization relative to rats.
Even though the use of fear conditioning has expanded exponentially in recent years, critical assumptions about baseline fear have remained untested and no standardized method of addressing differences in baseline fear has been developed. Researchers have used a variety of ways to report tone fear, making the results of many influential findings very difficult to interpret. A small selection of such examples include researchers that ignore baseline fear responses and do not report them (e.g. Gewirtz and Davis, 1997; Marsicano et al. 2002; Schafe et al. 2005; Han et al. 2007; Gogolla et al. 2009; Monfils et al. 2009), calculate tone fear by subtracting baseline freezing from tone freezing (e.g. Reijmers et al. 2007) or from inter-trial interval freezing (e.g. Huerta et al. 2000), or calculate tone fear using the Annau and Kamin (1961) suppression ratio (e.g. Shors et al. 2002; Bangasser et al. 2006). These concerns apply to virtually all fear conditioning methodologies, including fear potentiated startle, which uses a subtraction score based on the difference between baseline and cued responses (e.g. Gewirtz and Davis, 1997).
The purpose of the current study was to characterize the interaction between baseline and tone fear in mice, which have become an essential tool in current biomedical research. To do this, we conditioned two different levels of tone fear and then used post-training manipulations to produce a range of baseline freezing for the tone test. It should be noted that one assumption of our design, which is supported by the overall pattern of results, is that these post-training manipulations, such as extinction and additional US presentations did not influence the true level of tone fear. This approach provided a data set with quantitative gradations in both tone and baseline fear that could be used to assess the efficacy of four methods of reporting tone fear: absolute freezing level, ratio of tone to baseline freezing, subtraction of baseline freezing, and an adjusted score derived from using baseline freezing as a covariate in an analysis of covariance (ANCOVA).