The basolateral amygdala determines the effects of fear memory on sleep in an animal model of PTSD

Abstract

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.

Brief Optogenetic Inhibition of the Basolateral Amygdala (BLA) in Mice Alters Effects of Stressful Experiences on Rapid Eye Movement Sleep (REM)

“…For examples, the NMDA receptor antagonist, D,L-2-amino-5-phosphonovaleric acid (APV), infused into BLA prior to ST or prior to context reexposure blocked the acquisition and expression of freezing, respectively. 45,46 The results may appear to be inconsistent with our findings; however, we have found that inactivation of BLA with the GABA A agonist, muscimol, prior to ST in our paradigm blocks both freezing and fear-induced reductions in REM, 19 whereas inactivation after ST blocks only fear-conditioned reductions in REM. 47 Interestingly, post-ST administration of APV into BLA also does not block freezing.…”

“…However, the amygdala, which is essential for fear responses (reviewed in 16 ), also mediates the effects of stress and fearful memories on REM (reviewed in 17 ). Evidence from studies using localized pharmacological manipulations demonstrate roles for both the central (CNA) 18 and the basolateral (BLA) 19 nuclei of the amygdala in mediating these effects. BLA also appears critical for forming fear memories that can differentially impact sleep.…”

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

“…Our prior study inactivating BLA with Mus prior to ST also found an attenuation of freezing upon re-exposure to the fearful context (Wellman et al, 2014) whereas inactivating BLA after ST did not (Wellman et al, 2016). Previous work from other labs has also reported that inactivation of BLA prior to context re-exposure attenuates freezing in the fearful context (Helmstetter and Bellgowan, 1994; Muller et al, 1997).…”

“…Microinjections of the corticotropin releasing factor antagonist, antalarmin (ANT) into BLA of rats prior to shock training (ST) blocked both IS-induced reductions in REM sleep and the formation of memories that alter sleep without blocking fear memory as indicated by contextual freezing (Wellman et al, 2013). By comparison, global inactivation of BLA with microinjections of Mus, prior to ST blocked the post-training reduction in REM sleep seen in vehicle treated rats (Wellman, Fitzpatrick, Machida, and Sanford, 2014). Furthermore, in Mus treated rats, REM sleep after re-exposures to the fearful context was at baseline levels and freezing was significantly attenuated.…”

Effects of sleep on memory for conditioned fear and fear extinction.

“…Contextual and discrete cues associated with past fear conditioning can also reduce and fragment REM in the rat (Jha, Brennan, Pawlyk, Ross, & Morrison, 2005; Pawlyk et al, 2008; Sanford, Tang, Ross, & Morrison, 2003) for as long as 2 weeks post-conditioning (Pawlyk et al, 2008). The BLA has been identified as a site at which both acute stress and fear-conditioned stimuli exert their sleep-disruptive effects (Wellman, Fitzpatrick, Machida, & Sanford, 2014) and these effects have been linked to central actions of corticotropin releasing factor (CRF) (Liu et al, 2011; Wellman, Yang, Ambrozewicz, Machida, & Sanford, 2013; Yang, Tang, Wellman, Liu, & Sanford, 2009). Notably, fear-conditioning-induced sleep disruption can be ameliorated by extinction training (Wellman, Yang, Tang, & Sanford, 2008).…”