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

Abstract

Study objectives: To study long-term effects of conditioned fear on REM sleep (REMS) parameters in albino rats.

Design: We have investigated disturbances in sleep architecture, including muscle twitch density as REMS phasic activity, and freezing behavior in wakefulness, upon reexposure to a conditioned stimulus (CS) on Day 1 and Day 14 postconditioning.

Subjects: Male Sprague-Dawley rats prepared for polysomnographic recordings.

Interventions: After baseline sleep recording, the animals in the experimental group received five pairings of a 5-sec tone, co-terminating with a 1-sec, 1 mAfootshock. The control rats received similar numbers of tones and shocks, but explicitly unpaired. On postconditioning days, after reexposure to tones alone, sleep and freezing behavior were recorded.

Measurements and results: Conditioned fear significantly altered REMS microarchitecture (characterized as sequential-REMS [seq-REMS: < or =3 min episode separation] and single-REMS [sin-REMS: >3 min episode separation]) on Day 14. The total amount and number of seq-REMS episodes decreased, while the total amount and number of sin-REMS episodes increased. Further, the CS induced significant increases in freezing and REMS myoclonic twitch density in the experimental group. Reexposure to the CS produced no alterations in controls.

Conclusions: The results suggest that conditioned fear causes REMS alterations, including difficulty in initiating a REMS episode as indicated by the diminution in the number of seq-REMS episodes. Another finding, the increase in phasic activity, agrees with the inference from clinical investigations that retrieval of fearful memories can be associated with the long-term REMS disturbances characteristic of posttraumatic stress disorder.

Figure 1

Example of muscle twitch scoring. Depicted is the EMG channel from a period of REM sleep (REMS) preceded by NREM sleep (NREMS). We created two vertical lines (a and b) to demarcate the REMS period, and two horizontal lines (1 and 2) to capture twitches. The lower horizontal line (1) was set above the EKG captured by the EMG, and the upper horizontal line (2) was set to exclude obvious artifacts. In the section of REMS depicted, five twitches, two occurring close together, are evident.

Figure 2

Alterations in sequential REMS (seq-REMS) measures, single REMS (sin-REMS) measures and freezing (expressed as total time spent freezing/ total 5-min observation period x 100) upon reexposure to fearful stimuli on post-conditioning Day 1 and Day 14 in the FC and UP groups. Data is represented as mean + S.E.M. and shows a within group X days interaction. Significance level * P < 0.05, ** P < 0.01, *** P < 0.001

Figure 3

Significant correlations between [A] sin-REMS amount and % time spent freezing, [B] seq-REMS amount and % time spent freezing, at all time points, in the FC group. These measures were not correlated in the UP group [C and D].

Figure 4

Changes in myoclonic twitch measures during REMS and its correlation with % time spent freezing in the FC and UP groups. Total number of muscle twitches during REMS [A]; and number of muscle twitches/REMS episode [B] increased significantly (compared to baseline) in the FC group. There was a significant positive correlation between the total number of muscle twitches during REMS and % time spent freezing, in the FC group [C] but not in the UP group [D]. Significance level * P < 0.05