Stress-Enhanced Fear Learning, a Robust Rodent Model of Post-Traumatic Stress Disorder

Here we describe the detailed methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of post-traumatic stress disorder, in both rats and mice. The model utilizes aspects of Pavlovian fear conditioning and freezing as an index of enhanced fear in rodents.

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders such as post-traumatic stress disorder (PTSD). To understand the biological mechanisms of fear dysregulation in PTSD, it is important to start with a valid animal model of the disorder. This protocol describes the methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of PTSD, in both rats and mice. SEFL was developed to recapitulate critical aspects of PTSD, including long-term sensitization of fear learning caused by an acute stressor. SEFL uses aspects of Pavlovian fear conditioning but produces a distinct and robust sensitized fear response far greater than normal conditional fear responses. The trauma procedure involves placing a rodent in a conditioning chamber and administering 15 unsignaled shocks randomly distributed over 90 minutes (for rat experiments; for mouse experiments, 10 unsignaled shocks randomly distributed over 60 minutes are used). On day 2, rodents are placed in a novel conditioning context where they receive a single shock; then, on day 3 they are placed back in the same context as on day 2 and tested for changes in freezing levels. Rodents that previously received the trauma display enhanced levels of freezing on the test day compared to those that received no shocks on the first day. Thus, with this model, a single highly stressful experience (the trauma) produces extreme fear of the stimuli associated with the traumatic event.

Fear is a critical behavior for survival, enabling individuals to recognize and respond to threats. However, exaggerated fear responses can contribute to the development of psychiatric disorders such as post-traumatic stress disorder (PTSD). One characteristic of PTSD is an exaggerated response to mild stressors, particularly those reminiscent of the original trauma, and a tendency to develop new fears^1,2. In the laboratory, fear is often measured through freezing behavior, which is a reliable and ethologically valid index of fear in humans and rodents^3,4

1. Subjects

1. Rats
   1. Order rats to arrive when they are approximately 90 days old and single-housed in standard rat cages.
      Note: Single housing is advised, as group housing produces variability due to interactions between animals in the home cage, particularly following stress exposure. SEFL has been demonstrated in male and female rats, in Long-Evans and Sprague Dawley rats, and in rats as young as 19 days old^7,10.
   2. Randomly assign animals to at least two conditions: trauma (n = 8) and no trauma (n = 8) (see Rau et al.⁵ for additional cont

Results of the trauma context test on Day 2 are shown in Figure 1. Animals in the trauma condition showed significantly higher levels of freezing in Context A compared to the no trauma controls, indicating acquisition of fear to the trauma context [rats: F(1,17) = 23.58, p < 0.01; mice: F(1,14) = 666.50, p < 0.0001]. Freezing during the baseline period before the single shock in the novel context on Day 3 is shown in

SEFL is a robust behavioral model of PTSD that can be recapitulated in both rats and mice and can be used to study the sensitized fear responses that characterize PTSD. Following traumatic stress, rodents show an increased fear response in a distinctly different context only after that context is paired with a mild stressor that serves as a reminder of a previous traumatic experience. Following the traumatic stress rodents unsurprisingly show high levels of fear when returned to the traumatic stress context on Day 2, ind...

Dr. Fanselow is a founding board member of Neurovation Labs.

This work was funded by National Institute of Health R01AA026530 (MSF), Staglin Center for Brain and Behavioral Health (MSF), NRSA-F32 MH10721201A1 and NARSAD 26612 (AKR), and NSF DGE-1650604 (SG).