A Novel Animal Behavioral Model For Assessing Arousal and Anxiety States: Implications for Post-Traumatic Stress Disorder

Background: Post-traumatic stress disorder (PTSD) is a highly prevalent disorder with widespread impact on health co-morbidities, including cardiovascular disease (CVD). There is a pressing need to understand its underlying mechanisms. This study builds upon our previous published clinical and pre-clinical findings assessing cardiovascular and autonomic responses to stress. Using a recently developed behavioral model and lab-developed software for integrated behavioral and cardio-autonomic analysis, we sought to assess the impact of chronic unpredictable threat reminders on arousal and anxiety behavior as well as cardiovascular and autonomic function in pair-housed mice. Methods: Male and female C57bl/6J mice underwent Pavlovian auditory fear conditioning (n=10-16/group) and were returned to the home cage, equipped with speaker and top-down camera with infrared capabilities. Using a customized software to remotely deliver the conditioned stimulus (CS), pair-housed mice received 6 tones (30sec. 6kHz) throughout the light-dark (LD) cycle over a 24hr period (3 light, 3 dark), repeated for 14 days. The first CS during each LD cycle was recorded and freezing behavior was manually scored. Videos were used to train DeepLabCut TM (DLC) for automated behavioral analysis. On day 15, to evaluate whether evoked freezing is context-dependent, mice were placed into a novel context and CS-dependent freezing was measured. Mice then underwent elevated plus maze (EPM), uncued startle reactivity testing, and fear-potentiated startle (FPS) to assess locomotor, anxiety, and hyperarousal and heightened vigilant behaviors. Results: Following 14 days of chronic unpredictable conditioned threat reminders, fear-conditioned male and female mice exhibited strong CS-evoked freezing in the home cage. Circadian evaluation revealed enhanced freezing in the light versus the dark phase ( males, p<0.01; females, p<0.01). DLC analysis successfully and accurately quantified freezing of group-housed mice. Over 14 days, CS-dependent freezing declined, though this was context-dependent as freezing was significantly higher in a novel context on day 15 ( males, p<0.01; females, p=0.02). While chronic threat reminders did not affect behavior in the EPM, males, but not females, displayed hyperarousal in both uncued (startle to 120dB, males, p<0.01, females, p=0.62) and cued (FPS, males, p=0.04, females, p=0.60) startle testing, though neither males nor females had increased background anxiety during FPS ( males, p=0.77, females, p=0.77). Conclusions: Here we demonstrate, using lab-developed software, environment- and circadian-dependent conditioned freezing in the home cage of group-housed mice. These results further demonstrate that the delivery of chronic intermittent unpredictable conditioned threat reminders leads to an enhanced startle phenotype, that may be sex-dependent. Future studies are needed to assess direct measures of autonomic function in this model, which could provide novel insights into and potentially revealing new therapeutic and preventive strategies for mitigating PTSD-related autonomic dysfunction. (CDMRP) PR210574. This is the full abstract presented at the American Physiology Summit 2024 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.