A novel automated software and video analysis system for quantifying physiological responses of conditioned fear in mice
Authors: Smith Hannah , Turley Benjamin , Swiercz Adam , Yu Zhe , Marvar Paul 
Published: May 2023

BACKGROUND: Conditioned fear is an aversive learning process that is highly sensitive to sensory conditions such as environmental context, threat intensity and circadian rhythms. To better understand these external variables that impact conditioned fear learning, we developed a novel software application that allows for programmable remote conditioned stimulus (CS, ie., auditory tone) delivery and recording of group-housed mice in a homecage, or familiar environment. The goal of our study was therefore to leverage this technology to examine conditioned fear in the homecage across light-dark (LD) circadian cycles. METHODS: Male C57bl/6J mice underwent Pavlovian auditory fear conditioning (20 CS/US pairings), with a CS-only group as a control (n=4/group). Mice were then returned to the homecage, equipped with speaker and camera. Using a customized open-source software to remotely evoke an enhanced physiological state of fear in the homecage, mice received a select number of conditioned auditory stimuli (30sec. intermittent tones) throughout the LD cycle over 14 days. The software was configured to deliver six tones (6kHz) over a 24hr period (3 light, 3 dark). The first CS during each LD cycle was recorded and percent freezing behavior was manually scored. On day 15, to evaluate whether the HC-specific evoked fear response transfers to a new environment, mice were placed into a novel context and CS-dependent freezing was measured. RESULTS: Over 14 days, compared to controls, fear-conditioned pair-housed mice exhibited strong CS-evoked freezing in the homecage ( total freezing: FC 70.46%, CS-only 3.69%, F(2.29, 11.14)=10.78, p<0.01). Interestingly, when we evaluated CS-specific circadian responses, fear-conditioned mice exhibited enhanced freezing in the light versus the dark phase ( total freezing: light 84.56%, dark 56.36%, F(2.15, 6.46)=11.04, p<0.01). In fear-conditioned mice, there was a 36% reduction in total CS-dependent homecage freezing over 14 days. However, this was a context-dependent effect, as the reduction in freezing on day 14 was not observed in the novel context on day 15 ( t(3)=3.16, p=0.05). CONCLUSIONS: Here we demonstrate for the first time, using an open-source software developed in our laboratory, environment and circadian-dependent conditioned freezing responses in the homecage of group-housed mice. This study provides a proof-of-principle innovative approach for examining physiological outcomes of conditioned fear across ethologically relevant environments, which may help further the understanding for heightened physiological responses in neuropsychiatric diseases such as posttraumatic stress disorder (PTSD). To further elucidate impaired physiological mechanisms of disordered fear, future studies will integrate multimodal behavioral (DeepLabCutTM) and cardiovascular telemetry-based approaches. Congressionally Directed Medical Research Programs (CDMRP) PR210574 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.