Darting across space and time: Parametric modulators of sex-biased conditioned fear responses

Abstract

Pavlovian fear conditioning is a widely used behavioral paradigm for studying associative learning in rodents. Despite early recognition that subjects may engage in a variety of behaviors that reflect the experimental parameters of a given protocol, the last several decades have seen the field narrow its focus to measure freezing as the sole indicator of conditioned fear. Additionally, unconditioned responses such as shock-related activity are rarely considered. We previously reported that female Sprague Dawley rats are more likely than males to engage in darting, an escape-like conditioned response that is associated with heightened shock reactivity, but we did not establish whether darting was sensitive to manipulations of factors such as chamber size, shock intensity, or number of trials. Our goal here was to address these questions by defining parametric and phenotypic predictors of darting in both sexes. To better capture fear-related behavioral repertoires in our animals, we developed ScaredyRat, a custom Python tool that analyzes Noldus Ethovision-generated raw data files to identify Darters and quantify both conditioned and unconditioned responses. We find that like freezing, darting probability scales with experimental alterations in multiple dimensions. In most cases, the sex bias towards females persists, but males will transition to darting in extended, or overtraining fear conditioning protocols.

Conditional and unconditional components of aversively motivated freezing, flight and darting in mice

“… Fadok et al, 2017 used a unique two-component serial CS consisting of a 10 s tone followed immediately by a 10 s white noise ending with a 1 s shock and found that the initial component (tone) produced freezing, while the second component (noise) produced bursts of locomotion and jumping in mice. Gruene et al, 2015 reported that in rats a tone CS resulted in a similar burst of locomotion, labeled darting, and this was replicated in subsequent studies ( Colom-Lapetina et al, 2019 ; Mitchell et al, 2021 ). The results were interpreted as a competition between ‘active’ and ‘passive’ defenses.…”

“…We conjecture that the movements seen by Totty et al, 2021 exclusively in animals that were conditioned were largely this type of movement. Further, it is likely that the flight behavior in rats observed by other groups ( Colom-Lapetina et al, 2019 ; Gruene et al, 2015 ; Mitchell et al, 2021 ) is this type of movement as Mitchell et al report that the majority of their CS-elicited darting occurs late in the CS presentation, about 7–10 s into a 30 s tone CS.…”

“…Perhaps initial reports of sex differences with more frequent darting in females could be explained by differences in handling and stress provided to females as a result of monitoring estrous phase, a potentially stressful procedure for the animals for which there is not an ideal control in males. However, results were recently replicated in which females show more darting than males in a sample of animals for which estrous cycle was not monitored ( Mitchell et al, 2021 ). It is worth noting that Gruene et al, 2015 , Colom-Lapetina et al, 2019 , and Mitchell et al, 2021 , generally find very low levels of darting, with a majority of animals classified as nondarters.…”

“…Experimenters that have examined running-like locomotion in fear conditioning situations tend to collapse the behaviors under a single label such as darting, flight, or escape ( Colom-Lapetina et al, 2019 ; Fadok et al, 2017 ; Gruene et al, 2015 ; Mitchell et al, 2021 ; Totty et al, 2021 ). These behaviors are then thought of as ‘active’ behaviors that compete with a ‘passive’ freezing response ( Fadok et al, 2017 ; Gozzi et al, 2010 ; Gruene et al, 2015 ).…”

“…Initial reports suggest a sex difference in this noise-elicited flight behavior such that female rats show more of this behavior than males ( Gruene et al, 2015 ; Mitchell et al, 2021 ). Within each experiment, we found no such sex differences between male and female mice for the PAR and darting measures of flight behavior, and Totty et al, 2021 similarly found no sex differences in such behavior in male and female rats.…”