Long-term effect of cued fear conditioning on REM sleep microarchitecture in rats.

LEARNING TO ASSOCIATE AVERSIVE EVENTS WITH ENVIRONMENTAL STIMULI THAT RELIABLY PREDICT THESE EVENTS IS INDISPENSABLE TO THE SURVIVAL of organisms. Pavlovian fear conditioning is an example of such learning in which an emotionally neutral conditioned stimulus (CS), such as a tone, is paired with an aversive, unconditioned stimulus (US), usually electrical footshock.1 The CS, by virtue of its relationship with the US, acquires aversive properties and elicits responses normally induced by threatening stimuli, such as freezing and ultrasonic vocalizations.2 This paradigm has been used in numerous studies to explore mechanisms that may underlie the psychophysiological alterations associated with posttraumatic stress disorder (PTSD). Experiencing traumatic and aversive events in humans can induce several long-term behavioral and physiological disturbances, including changes in sleep.3,4 Sleep alterations also result from exposure to fear conditioning in both rats5–7 and mice.8,9 One observes changes in vigilance states characterized predominantly by decreased REM sleep (REMS), while NREM sleep (NREMS) remains unaffected.5,6,8 Although the effects of fear conditioning on REMS can be assessed by looking at REMS in toto, various stressors can also alter its microarchitectural pattern (i.e., parcellation of REMS into sequential and single episodes) in rats.10 REMS can be characterized as sequential-REMS (seq-REMS) (comprised of REMS episodes separated by ≤ 3 min intervals, which appear in clusters), and single-REMS (sin-REMS) (with episodes preceded and followed by >3 min intervals).10 This distinction is important because stressors such as low ambient temperature and immobilization have differential effects on sin-REMS and seq-REMS.10,11 For example, rats placed in a cold environment show a reduction in REMS amount. During the recovery period at a normal room temperature, REMS is increased via an increase in the number of seq-REMS bouts. Because temperature is not tightly regulated during REMS due to an absence of hypothalamic control, Amici et al10 suggest that the brief intervals of wakefulness or NREMS punctuating a REMS cluster allow sufficient REMS to be made up by ensuring short periods of homeostatic regulation of the body during a cluster, and that this could not occur, were the REMS deficit to be made up by extended periods of sin-REMS. Similarly, after immobilization stress, the increase in REMS is associated with only a modest increase in sin-REMS, but a considerable increase in seq-REMS, which may result from recovery from sleep deprivation during the immobilization procedure.11 A bimodal distribution of REMS intervals also exists in other species, including humans.10 Altered sleep quality and/or quantity can itself be stressful to an organism and contribute to the emotional and behavioral disturbances symptomatic of psychiatric disorders. We have reported that exposure to fear-inducing tones (the CS), previously paired with brief, mild electrical footshock, alters both REMS and its microarchitecture,5 but the persistence of these effects over time has received less research attention.8 Interestingly, there is often a progressive increase in memory strength over time following an aversive stimulus (measured as a change in the magnitude of the freezing response), without further exposure to the stimulus itself: this has been referred to as an incubation process.12 Therefore, in this study, we evaluated the impact of reexposure to fearful cues on the sleep architecture and REMS microarchitecture of rats, both 24 hr (short-term effect, Day 1) and two weeks (long-term effect, Day 14) after conditioning. In addition, we examined the effects of reexposure to the CS on a more common behavioral index of anxiety, freezing. We and others have reported earlier that military veterans with PTSD have increased phasic muscle activity during REMS13–15; hence, we also evaluated the frequency of myoclonic twitches as a measure of phasic REMS events. Several studies suggest that fear conditioning alters the amount and microarchitecture of REMS, but does not alter the sleep efficiency or NREMS amount.5,6,8,16 Hence, in this study we hypothesized that reexposure to cues associated with footshock should alter REMS microarchitecture, primarily by changing the sin-REMS, seq-REMS, and cluster amount. We also expected that fear conditioning would increase the number of myoclonic twitches during REMS. Finally, we hypothesized that changes in REMS microarchitecture and myoclonic twitches are also due to conditioned anxiety, and as such should correlate with freezing, a well-established indicator of conditioned anxiety.