Predicting stress resilience and vulnerability: brain-derived neurotrophic factor and rapid eye movement sleep as potential biomarkers of individual stress responses.

STUDY OBJECTIVES: To examine the rapid eye movement sleep (REM) response to mild stress as a predictor of the REM response to intense stress and brain-derived neurotrophic factor (BDNF) as a potential biomarker of stress resilience and vulnerability. METHODS: Outbred Wistar rats were surgically implanted with electrodes for recording electroencephalography (EEG) and electromyogram (EMG) and intraperitoneal Data loggers to record body temperature. Blood was also obtained to measure circulating BDNF. After recovery, rats were exposed to mild stress (novel chamber, NC) and later intense stress (shock training, ST), followed by sleep recording. Subsequently, rats were separated into resilient (Res; n=27) or vulnerable (Vul; n = 15) based on whether or not there was a 50% or greater decrease in REM after ST compared to baseline. We then compared sleep, freezing, and the stress response (stress-induced hyperthermia, SIH) across groups to determine the effects of mild and intense stress to determine if BDNF was predictive of the REM response. RESULTS: REM totals in the first 4 hours of sleep after exposure to NC predicted REM responses following ST with resilient animals having higher REM and vulnerable animals having lower REM. Resilient rats had significantly higher baseline peripheral BDNF compared to vulnerable rats. CONCLUSIONS: These results show that outbred rats display significant differences in post-stress sleep and peripheral BDNF identifying these factors as potential markers of resilience and vulnerability prior to traumatic stress.

Differential behavioral, stress, and sleep responses in mice with different delays of fear extinction.

STUDY OBJECTIVES: Sleep, in particular rapid eye movement (REM), has been linked to fear learning and extinction; however, their relationship is poorly understood. We determined how different delays of extinction training (ET) impact fear-conditioned behaviors, changes in sleep, and stress responses. METHODS: EEG activity, movement, and body temperature in mice were monitored via telemetry. Following contextual fear conditioning (shock training [ST]), separate groups of mice were reexposed to the context at 24-hour post-ST (24h ET-1) and at 48-hour post-ST (48h ET-1). Post-ET sleep amount and sleep-associated EEG (delta and theta) activity were compared to baseline and to post-ST sleep. Freezing, locomotion, grooming, and rearing were monitored to determine effects of ET on fear behaviors. Body temperature immediately after ET was monitored to assess stress-induced hyperthermia (SIH). RESULTS: 24h ET-1 and 48h ET-1 produced similar freezing and REM reductions, but dissimilar rearing activity and SIH. 24h ET-1 was followed by periods of suppressed REM-associated theta (REM-θ) activity, immediately after ET and during the subsequent dark period. Suppressed REM-θ was specific to sleep after 24h ET-1, and did not occur after ST, nor after 48h ET-1. CONCLUSIONS: ET-1 at 24 and 48 hours after ST was associated with similar freezing and REM amounts, but with differences in other overt behaviors, in REM-θ, and in SIH. Freezing was not predictive of changes in other fear-associated responses. This study demonstrated that consideration of time delay from fear acquisition to extinction is important when assessing the relationships between extinction and behavior, sleep, and stress responses.

Antagonism of corticotropin releasing factor in the basolateral amygdala of resilient and vulnerable rats: Effects on fear-conditioned sleep, temperature and freezing.

The basolateral nucleus of the amygdala (BLA) plays a significant role in mediating individual differences in the effects of fear memory on sleep. Here, we assessed the effects of antagonizing corticotropin releasing factor receptor 1 (CRFR1) after shock training (ST) on fear-conditioned behaviors and sleep. Outbred Wistar rats were surgically implanted with electrodes for recording EEG and EMG and with bilateral guide cannulae directed at BLA. Data loggers were placed intraperitoneally to record core body temperature. The CRFR1 antagonist, antalarmin (ANT; 4.82 mM) was microinjected into BLA after shock training (ST: 20 footshocks, 0.8 mA, 0.5 s duration, 60 s interstimulus interval), and the effects on sleep, freezing and the stress response (stress-induced hyperthermia, SIH) were examined after ST and fearful context re-exposure alone at 7 days (CTX1) and 21 days (CTX2) post-ST. EEG and EMG recordings were scored for non-rapid eye movement sleep (NREM), rapid eye movement sleep (REM) and wakefulness. The rats were separated into 4 groups: Vehicle-vulnerable (Veh-Vul; n = 10), Veh-resilient (Veh-Res; n = 11), ANT-vulnerable (ANT-Vul; n = 8) and ANT-resilient (ANT-Res; n = 8) based on whether, compared to baseline, the rats showed a decrease or no change/increase in REM during the first 4 h following ST. Post-ST ANT microinjected into BLA attenuated the fear-conditioned reduction in REM in ANT-Vul rats on CTX1, but did not significantly alter REM in ANT-Res rats. However, compared to Veh treated rats, REM was reduced in ANT treated rats on CTX2. There were no group differences in freezing or SIH across conditions. Therefore, CRFR1 in BLA plays a role in mediating individual differences in sleep responses to stress and in the extinction of fear conditioned changes in sleep.

The basolateral amygdala can mediate the effects of fear memory on sleep independently of fear behavior and the peripheral stress response.

Fear conditioning associated with inescapable shock training (ST) and fearful context re-exposure (CR) alone can produce significant behavioral fear, a stress response and alterations in subsequent REM sleep. These alterations may vary among animals and are mediated by the basolateral nucleus of the amygdala (BLA). Here, we used the GABAA agonist, muscimol (Mus), to inactivate BLA prior to CR and examined the effects on sleep, freezing and stress-induced hyperthermia (SIH). Wistar rats (n=28) were implanted with electrodes for recording sleep, data loggers for recording core body temperature, and with cannulae aimed bilaterally into BLA. After recovery, the animals were habituated to the injection procedure and baseline sleep was recorded. On experimental day 1, rats received ST (20 footshocks, 0.8mA, 0.5s duration, 60s interstimulus interval). On experimental day 7, the rats received microinjections (0.5µl) into BLA of either Mus (1.0µM; n=13) or vehicle (Veh; n=15) prior to CR (CR1). On experimental day 21, the animals experienced a second CR (CR2) without Mus. For analysis, the rats were separated into 4 groups: (Veh-vulnerable (Veh-Vul; n=8), Veh-resilient (Veh-Res; n=7), Mus-vulnerable (Mus-Vul; n=7), and Mus-resilient (Mus-Res; n=6)) based on whether or not REM was decreased, compared to baseline, during the first 4h following ST. Pre-CR1 inactivation of BLA did not alter freezing or SIH, but did block the reduction in REM in the Mus-Vul group compared to the Veh-Vul group. These data indicate that BLA is an important region for mediating the effects of fearful memories on sleep.

Individual Differences in Animal Stress Models: Considering Resilience, Vulnerability, and the Amygdala in Mediating the Effects of Stress and Conditioned Fear on Sleep.

STUDY OBJECTIVES: To examine the REM sleep response to stress and fearful memories as a potential marker of stress resilience and vulnerability and to assess the role of the basolateral amygdala (BLA) in mediating the effects of fear memory on sleep. METHODS: Outbred Wistar rats were surgically implanted with electrodes for recording EEG and EMG and with bilateral guide cannulae directed at the BLA. Data loggers were placed intraperitoneally to record core body temperature. After recovery from surgery, the rats received shock training (ST: 20 footshocks, 0.8 mA, 0.5-s duration, 60-s interstimulus interval) and afterwards received microinjections of the GABAA agonist muscimol (MUS; 1.0 µM) to inactivate BLA or microinjections of vehicle (VEH) alone. Subsequently, the rats were separated into 4 groups (VEH-vulnerable (VEH-Vul; n = 14), VEH-resilient (VEH-Res; n = 13), MUS-vulnerable (MUS-Vul; n = 8), and MUS-resilient (MUS-Res; n = 11) based on whether or not REM was decreased, compared to baseline, during the first 4 h following ST. We then compared sleep, freezing, and the stress response (stress-induced hyperthermia, SIH) across groups to determine the effects of ST and fearful context re-exposure alone (CTX). RESULTS: REM was significantly reduced on the ST day in both VEH-Vul and MUS-Vul rats; however, post-ST MUS blocked the reduction in REM on the CTX day in the MUS-Vul group. The VEH-Res and MUS-Res rats showed similar levels of REM on both ST and CTX days. The effects of post-ST inactivation of BLA on freezing and SIH were minimal. CONCLUSIONS: Outbred Wistar rats can show significant individual differences in the effects of stress on REM that are mediated by BLA. These differences in REM can be independent of behavioral fear and the peripheral stress response, and may be an important biomarker of stress resilience and vulnerability.