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.

Fear conditioning (inescapable shock training (ST)) and fearful context re-exposure (CR) alone can produce significant fear indicated by increased freezing and reductions in subsequent REM sleep. Damage to or inactivation of the basolateral nucleus of the amygdala (BLA) prior to or after ST or prior to CR generally has been found to attenuate freezing in the shock training context. However, no one has examined the impact of BLA inactivation on fear-induced changes in sleep. Here, we used the GABAA agonist, muscimol (MUS), to inactivate BLA prior to ST, the period when fear is learned, and assessed sleep after ST and sleep and freezing after two CR sessions. Wistar rats (n=14) were implanted with electrodes for recording sleep and with cannulae aimed bilaterally into BLA. After recovery, the animals were habituated to the injection procedure (handling) over 2 consecutive days and baseline sleep following handling was recorded. On experimental day 1, the rats were injected (0.5ul) into BLA with either MUS (1.0uM; n = 7) or vehicle (distilled water (dW), n = 7) 30 min prior to ST (20 footshocks, 0.8mA, 0.5s duration, 60s interstimulus interval). On experimental days 7 and 21, the animals experienced CR (CR1 and CR2, respectively) alone. EEG and EMG were recorded for 8h on each day and the recording was scored for non-rapid eye movement sleep (NREM), rapid eye movement sleep (REM), and wakefulness. Freezing was examined during CR1 and CR2. MUS microinjections into BLA prior to ST blocked the post-training reduction in REM sleep seen in vehicle treated rats. Furthermore, in MUS treated rats, REM sleep after CR1 and CR2 was at baseline levels and freezing was significantly attenuated. Thus, BLA inactivation prior to ST blocks the effects of footshock stress on sleep and reduces fear memory, as indicated by the lack of freezing and changes in sleep after CR. These data indicate that BLA is an important regulator of stress-induced alterations in sleep, and an important site for forming fear memories that can alter sleep.

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 (0.5ul) into BLA of either Mus (1.0uM; 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 4 h 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.

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.