Research report
Sleep after differing amounts of conditioned fear training in BALB/cJ mice

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Abstract

Shock training and auditory cues associated with shock produce alterations in sleep that can be long-lasting in BALB/cJ (C) mice. We examined sleep in C mice after different amounts of shock training, and after cues with different strength cue-shock associations. Mice were implanted with transmitters for determining sleep via telemetry. After baseline sleep recording, the mice were trained (between 08:00 and 09:00 h) to associate a cue (tone) with footshock in either single shock training (SST: a single tone-shock pairing) or multiple shock training (MST: 15 tone-shock pairings) conditions. For testing, the mice were presented 15 cues (tone only) in their home cage between 10:45 and 11:00 h on post-training days 6, 13, 20, 27, and 34 (Cue 1 to Cue 5) following shock training. Sleep was recorded for two days after shock training or cue presentation. A separate group of mice received 15 tone-shock pairings and had their sleep recorded for 10 consecutive uninterrupted days. Both SST and MST mice showed decreases in rapid eye movement sleep (REM) after shock training, with the larger effect in the MST mice. Only MST mice showed significant reductions in REM in response to the fearful cues, and longer-term alterations in sleep could be observed even on the day after cue presentation. These results indicate that reminders of an aversive event can impact sleep for prolonged periods, and that the degree of the impact varies with amount of training.

Introduction

Conditioned fear is a classical conditioning procedure in which an association is made between a neutral stimulus (generally visual or auditory) or context and an aversive stimulus (usually footshock) [9], [10]. Through training, the conditioned cue or context acquires fear-inducing qualities and produces behavioral and physiological responses that share features with generalized anxiety disorder in humans [9], [10]. The immediate responses to fearful stimuli such as freezing (e.g. [3], [17], [18]), autonomic responses (e.g. [13], [16]) and fear-potentiated startle (e.g. [9], [10]) have received considerable attention by researchers, whereas longer-term, and potentially more lasting effects have received less consideration. However, the alterations in behavior that occur in the aftermath of various stressors suggest that examining the long-term effects of a fearful experience will be important for fully understanding the genesis of affective disorders (e.g. [1], [30], [31]).

We have demonstrated that the shock training phase of fear conditioning, and the presentation of fearful cues and re-exposure to shock contexts can alter sleep architecture in mice [24], [25] and rats [23]. In these studies, we employed shock training sessions on four consecutive days to ensure that the cue-shock association had been made. Changes were always observed in rapid eye movement sleep (REM), though alterations in other states occurred in some mouse strains. These studies demonstrated that mice that showed greater reactivity or “anxiousness” on putative behavioral tests of anxiety were more susceptible to alterations in sleep after shock training and after the presentation of fear-conditioned cues [24]. They also demonstrated that sleep could be disturbed for an extended period after the presentation of shock or fearful cues.

Stressful experiences can lead to enduring alterations in emotion and behavior in humans [26] and animals [1], [19], [30], [31]. Disturbances in sleep also often follow a stressful or traumatic event (reviewed in [14]), and the persistence of these disturbances may be predictive for future psychiatric and physical pathology [12], [14]. Thus, the alterations in sleep produced by fear conditioning, and the potential long-term effects of conditioning processes raise the question of whether fear-conditioned cues could influence sleep over an extended period of time. Also, the impact of gradations in the severity of the shock stressor, and the potential differences in the immediate and longer-term effects of different levels of stress have not been examined with respect to sleep. Therefore, in this study, we gave mice a single day of training with either 1 or 15 tone-shock pairings, and evaluated the impact of the presentation of fear-conditioned tones on sleep over the course of a month after training. We conducted the experiment using BALB/cJ (C) mice, one of the strains that we have studied that is highly reactive on behavioral tests of anxiety [24], [25], [28].

Section snippets

Subjects

The subjects were 23 male C mice (Jackson Laboratories, Bar Harbor Maine) weighing 20–25 g at the beginning of the experiment. Food and water were available ad libitum. The recording room was kept on a 12:12 light:dark cycle with lights on from 07:00 to 19:00 h. Ambient temperature was maintained at 24.5±0.5 °C.

Surgery

The mice were surgically implanted with telemetry transmitters (DataSciences ETA10-F20) for recording electroencephalogram (EEG) and movement. All surgical procedures were conducted with

Sleep architecture after shock training

Fig. 1 presents total NREM and REM for the SST and MST groups plotted hourly on the day of shock training (Day 1), and on the next full 24 h period (Day 2). Comparisons were made to plots of uninterrupted baseline recordings. These plots demonstrate the impact that shock training can have across days, and indicate that even a single shock training session can influence sleep, though the impact may be less than that seen with multiple shocks.

Sleep across blocks

Sleep after shock training was compared to sleep during

Sleep after shock training

One of the primary findings of this study was that the impact of shock training on sleep varied with the amount of shock each animal received. The results indicate that as little as a single footshock can produce significant immediate reductions in NREM and REM sleep in the reactive C strain of mice. The impact of fifteen tone-shock pairings was considerably greater, producing both immediate alterations in sleep, and longer-term alterations that could be observed on the day following the shock

Acknowledgements

We would like to thank Ms. Linghui Yang and Mr. Stuart Orchard for technical assistance in conducting this study. This work was supported by MH61716.

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