The anterior cingulate cortex and its role in controlling contextual fear memory to predatory threats

Predator exposure is a life-threatening experience and elicits learned fear responses to the context in which the predator was encountered. The anterior cingulate area (ACA) occupies a pivotal position in a cortical network responsive to predatory threats, and it exerts a critical role in processing fear memory. The experiments were made in mice and revealed that the ACA is involved in both the acquisition and expression of contextual fear to predatory threat. Overall, the ACA can provide predictive relationships between the context and the predator threat and influences fear memory acquisition through projections to the basolateral amygdala and perirhinal region and the expression of contextual fear through projections to the dorsolateral periaqueductal gray. Our results expand previous studies based on classical fear conditioning and open interesting perspectives for understanding how the ACA is involved in processing contextual fear memory to ethologic threatening conditions that entrain specific medial hypothalamic fear circuits.


Sample-size estimation
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Replicates
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We first run the pharmacogenetic inhibition of the ACA followed by the functional tracing of the ACA inputs during the acquisition and expression of contextual fear. Next, we examined how silencing the AM > ACA pathway influences contextual fear acquisition to the predatory threat. In the following experiments, we silenced the ACA projections to different targets and examined how it influences the acquisition and/or expression of contextual fear. The experiments were not replicated and followed the sequence of the findings. There was no outlier exclusion, and the criteria for including the experiments were based on the correct position of viral transfection and optic fiber placement, as stated in the manuscript (see Methods page 30 lines 7 and 32)

Statistical reporting
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Additional data files ("source data")
• We encourage you to upload relevant additional data files, such as numerical data that are represented as a graph in a figure, or as a summary table • Where provided, these should be in the most useful format, and they can be uploaded as "Source data" files linked to a main figure or table • Include model definition files including the full list of parameters used • Include code used for data analysis (e.g., R, MatLab) • Avoid stating that data files are "available upon request" Statistical analysis methods were fully described in the Methods Section (see page 34 line 8 and 29). The raw data for the behavior experiments are plotted in the figure graphs, indicating the mean (± SEM) values. Complete statistical analyses for behavioral experiments are described in the Figure Legends, and the statistical analyses for the cell counting can be found in the Results Section. In both instances, exact values of N, means, SEM, and effect sizes are fully reported, and exact p-values whenever possible for significant and nonsignificant results.
The animals were randomly allocated to each experimental group without utilizing a restricted randomization criterium. As indicated in the manuscript, behavioral sessions were blindly scored by a trained observer (see Methods page 29 line 14), and the statistical analyses were conducted by an experimenter (MVCB) without previous knowledge of the experimental results (see Methods page 35 line 8).