# Behavioral phenotype analysis highlights sleep heterogeneity and brain cellular activations in a mouse model of PTSD

## TL;DR

This study identifies different behavioral and sleep patterns in mice after stress, linking them to brain activity and resilience to PTSD-like symptoms.

## Contribution

The study introduces a two-step behavioral scoring system to identify distinct PTSD-like phenotypes in mice and links them to REM sleep and brain activation patterns.

## Key findings

- Resilient mice spent more time in REM sleep compared to susceptible mice.
- Behavioral alterations correlated with c-Fos activation in specific amygdala regions.
- Two susceptible phenotypes (freezers and escapers) showed distinct defensive behaviors and brain activation patterns.

## Abstract

Exposure to intense and unavoidable stressor can lead to Post Traumatic Stress Disorder (PTSD) in some individuals. The heterogeneity of symptom expression, including sleep alterations, may explain the high rate of both non-responder and relapse of current treatments. Understanding individual-specific brain activity related to stress behavioral responses seems crucial for developing refined and personalized treatments strategies.

For this purpose, we evaluated the behavior of outbred mice through multiple tests up to 28 days after two inescapable electrical foot-shocks (FS). A two-step behavioral phenotype analysis successfully identified three phenotypes among FS mice. First, the scoring of behavioral alterations severity, based on PTSD-like symptoms, differentiated susceptible and resilient animals. Second, the specific type of stress-induced defensive behaviors further categorized two susceptible phenotypes: freezers and escapers.

Sleep patterns specific to phenotype emerged 14 days after the FS exposure. Notably, resilient mice exhibited more time spent in rapid eye movement sleep than susceptible animals, a variable that was negatively correlated to the behavioral alteration severity score.

Finally, behavioral profiling highlighted different c-Fos protein expressions in amygdala (AMG) and in the periaqueductal gray matter (PAG) across phenotypes, suggesting region-specific neural responses. Specifically, the severity of PTSD-like behaviors was correlated to the right lateral and central-lateral AMG cellular activations.

In conclusion, this study emphasizes the relevance of using composite score of multiple behavioral tests to better understand the complexity of stress responses and interindividual variability. Moreover, our findings suggest a role of REM sleep in promoting behavioral resilience to high-intensity stress.

Decoding behavioral heterogeneity after foot-shock stress: resilience and susceptible phenotypes linked to REM sleep and brain cellular activations.The analysis of behavioral heterogeneity in mice receiving two electrical foot-shocks (FS; 1.6 mA, 2 s) highlighted three phenotypes with specific rapid eye movement (REM) sleep pattern and brain cellular activation within key brain regions implicated in PTSD (Amygdala, AMG, and periaqueductal gray matter, PAG). This heterogeneity was assessed by scoring (from 0 to 3), for each item, the deviation from the control (non-stressed) group median dataset of two variables for each animal. Then, each item is illustrated by a composite score (from 0 to 6) of those two variables.Four behavioral tests were chosen to model the four main PTSD-like symptoms: intrusion was assessed through percentage of freezing and escaping when animals were exposed to a FS cue reminder; the avoidance was quantified by the time spent and the velocity of animal in open arms of an elevated-plus maze; the altered reactivity was represented by the percentage of freezing and escaping when animals were exposed to a new context and new object; eventually the altered arousal was assessed in this study through the amplitude of the maximum response and the latency to this response during an airpuff startle test.Sleep was recorded during 24 h, 14 days after the foot-shock exposure. The two sleep stages slow-wave sleep (SWS) and rapid eye movement sleep (REM) were quantified every 5 sec-epoch. Sleep entry scores were calculated as the sum the latency to first sleep latency score and number of sleep episodes score. Sleep continuity scores were calculated as the sum of the scores of the percentage of time spent and the mean duration of sleep episodes for each sleep stage. The cellular activation variability was quantified by scoring the c-Fos cells numbers in AMG nuclei and PAG subcolumns. Two scores were assessed for each structure. For the amygdala, it is represented by the Basolateral (BLA) score, composite score of both basoanterior and lateral amygdala cellular activation scores, and the Central (CeA) score, composite of the central lateral and central medial amygdala nuclei activation scores. Both dorsal PAG (dPAG) and ventral PAG (vPAG) scores, composite scores of the cellular activation of the anterior and posterior part each PAG subcolumn, expressed the cellular activation of the PAG.The present results support the existence of resilient and two distinct susceptible (PTSD-like) phenotypes following foot-shock stress in mice and suggest a potential role of REM sleep in behavioral resilience. This study also underscores the relevance of using a composite score derived from multiple behavioral tests to better characterize complex interindividual variability.Image 1

•The behavioral alteration severity score of foot-shocked mice reveals resilient and two susceptible phenotypes.•These phenotypes present different cellular activations in the amygdala and the periaqueductal gray matter.•The behavioral profiling highlights specific sleep patterns between both resilient and susceptible mice.•Behavioral alterations are correlated to both the activation of amygdala nuclei and rapid eye movement (REM) sleep.

The behavioral alteration severity score of foot-shocked mice reveals resilient and two susceptible phenotypes.

These phenotypes present different cellular activations in the amygdala and the periaqueductal gray matter.

The behavioral profiling highlights specific sleep patterns between both resilient and susceptible mice.

Behavioral alterations are correlated to both the activation of amygdala nuclei and rapid eye movement (REM) sleep.