Chronic stress and sex differences on the recall of fear conditioning and extinction

Abstract

Chronic stress effects and sex differences were examined on conditioned fear extinction. Male and female Sprague-Dawley rats were chronically stressed by restraint (6 h/d/21 d), conditioned to tone and footshock, followed by extinction after 1 h and 24 h delays. Chronic stress impaired the recall of fear extinction in males, as evidenced by high freezing to tone after the 24 h delay despite exposure to the previous 1 h delay extinction trials, and this effect was not due to ceiling effects from overtraining during conditioning. In contrast, chronic stress attenuated the recall of fear conditioning acquisition in females, regardless of exposure to the 1 h extinction exposure. Since freezing to tone was reinstated following unsignalled footshocks, the deficit in the stressed rats reflected impaired recall rather than impaired consolidation. Sex differences in fear conditioning and extinction were observed in nonstressed controls as well, with control females resisting extinction to tone. Analysis of contextual freezing showed that all groups (control, stress, male, female) increased freezing immediately after the first tone extinction trial, demonstrating contextual discrimination. These findings show that chronic stress and sex interact to influence fear conditioning, with chronic stress impairing the recall of delayed fear extinction in males to implicate the medial prefrontal cortex, disrupting the recall of the fear conditioning acquisition in females to implicate the amygdala, and nonstressed controls exhibiting sex differences in fear conditioning and extinction, which may involve the amygdala and/or corticosterone levels.

Figure 1

Percentage of freezing to tone during fear conditioning and extinction in male and females rats. A. Note the enhanced freezing to tone of the chronically stressed males during the first block of extinction on day 2, despite showing similar freezing to tone as control males during fear conditioning and extinction on day 1. B. When tone was paired with footshock, but no extinction trials were given on day 1, male rats (control and chronic stress) receiving extinction to tone on the second day maintained high and similar levels of freezing to tone. C. When tone and footshock were explicitly not paired (unpaired), freezing to tone decreased across blocks during extinction on day 1 for both control and chronically stressed males. Notably, the chronically stressed males showed freezing to tone near 20% on day 2, which never fell below 50% when tone and footshock were paired (compare to A and B). D. When tone was paired with footshock, chronically stressed female rats froze to tone significantly less than did the controls during extinction on day 1, and this pattern continued during extinction on day 2. E. When the tone was paired with footshock, but no extinction trials were given on day 1, chronically stressed females froze less to tone than did the controls. F. When tone and footshock were explicitly not paired (unpaired), female rats (control and chronic stress) froze less to tone than did rats in the paired conditioning paradigm (compared to D). Habit. = habituation trials; Cond. = conditioning trials; Extinct. = extinction trials. Data represent means ± S.E.M. * p < 0.05 compared to controls. Controls = black symbols; Chronic stress = white symbols. n = 8 to 10 rats/group.

Figure 2

Percentage of freezing to tones following two unsignalled footshocks. Male rats (control and chronic stress) and chronically stressed females froze more to tone following the two unsignalled footshocks than compared to their own level of freezing to tone at the last extinction trial. The control females failed to show enhanced freezing to tone following the unsignalled footshock, in part, because they maintained high freezing during extinction. Data represented as mean ± S.E.M. *p < 0.05 for reinstatement compared to the last extinction trial from the same group.

Figure 3

Percentage of freezing to context at designated periods during extinction to tone. A. Diagram outlining the different time points by which freezing to context was sampled. Triangles represent arbitrary data points of tone presentation. The boxed letter “A” represents the 30 seconds before the first tone (T1) presented during extinction on day 1. The boxed letters “B”, “C”, and “D” represent the 30 seconds immediately following the termination of T1, the last tone during extinction on day 1 (T15), and the first tone presentation on day 2 (T16), respectively. B. Chronically stressed rats (male and female) froze more to context than did the controls of both sexes (collapsed across contexts “A” and “B”). Once the first tone (T1) was presented, rats in all groups froze more to context B than to context A. C. As extinction trials progressed on day 1, control females significantly increased freezing to context, while the remaining rats (control and chronically stress males and chronically stressed females) froze similarly to context throughout extinction. Freezing to context was similar between all rats immediately prior to the extinction trials on day 2. Data represented as means ± S.E.M. * p < 0.05 for chronically stressed rats (male and female) compared to controls across contexts A and B. # p < 0.05 compared to freezing in context A for the same group.