Sex differences in fear regulation and reward-seeking behaviors in a fear-safety-reward discrimination task

Abstract

Reward availability and the potential for danger or safety potently regulate emotion. Despite women being more likely than men to develop emotion dysregulation disorders, there are comparatively few studies investigating fear, safety and reward regulation in females. Here, we show that female Long Evans rats did not suppress conditioned freezing in the presence of a safety cue, nor did they extinguish their freezing response, whereas males did both. Females were also more reward responsive during the reward cue until the first footshock exposure, at which point there were no sex differences in reward seeking to the reward cue. Darting analyses suggest females were able to regulate this behavior in response to the safety cue, suggesting they were able to discriminate between fear and safety cues but did not demonstrate this with conditioned suppression of freezing behavior. However, levels of darting in this study were too low to make any definitive conclusions. In summary, females showed a significantly different behavioral profile than males in a task that tested the ability to discriminate among fear, safety and reward cues. This paradigm offers a great opportunity to test for mechanisms that are generating these behavioral sex differences in learned safety and reward seeking.

Keywords: Extinction; Fear; Reward; Safety; Sex differences.

Figure 1.. Females spent more time reward seeking in response to the reward cue.

A) Schematic depicting experimental outline. During reward pre-training and habituation sessions, rats (16 males, 20 females) received 25 cue-sucrose pairings per session. B) Averaged percent time spent in the reward port during the five reward pre-training sessions (R1–5) and habituation session (HAB). Across all sessions females spent more time reward seeking than males. C) Averaged latency to enter the port after cue onset (in seconds). Across all sessions females were faster to enter the port than males. Means +/− SEM. **main effect of sex, p<0.01.

Figure 2.. Females did not show inhibition of conditioned freezing in the presence of the safety cue.

A) Schematic depicting experimental outline. During the 4 DC sessions, rats (16 males, 20 females) were presented with four types of cued trials: reward cue-sucrose, fear cue-shock, fear+safety cue with no footshock and the safety cue presented alone without footshock. B) Averaged percent time spent in the port during each cue across the 4 DC sessions. Both males and females showed significantly higher reward seeking during the reward cue compared to all other cues during every DC session. During DC1, females showed significantly higher reward seeking to the reward cue compared to males. C) Averaged percent time spent freezing during each cue across the 4 DC sessions. During DC3 and DC4, males showed significantly lower freezing to the fear+safety cue (and reward and safety cues) when compared to the fear cue. Females did not show significant inhibition of conditioned freezing to the fear+safety cue compared to the fear cue during any DC session. Females also showed significantly higher freezing to the fear+safety cue compared to males during DC2–4. D) Darting behavior during each cue across the 4 DC sessions. During DC4 females showed significantly more darts than males during the fear and fear+safety cues. Females also showed more darts during the fear cue than the fear+safety cue. Means +/− SEM. # p<0.05, ####p<0.0001 within sex, between cue comparison; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 within cue, between sex comparison. ^^^^main effect of cue, p<0.0001.

Figure 3.. Females did not show significant extinction of fear.

A) Schematic depicting experimental outline. During extinction training both the reward and fear cues were presented in the same session without sucrose or footshock. During the test for extinction memory 1 day later all cues were presented without sucrose or footshock. Bi) Averaged percent time spent in the port during each reward cue presentation during extinction training. No significant differences were found between males and females during extinction training. Bii) Averaged percent time spent in the port during each cue 1 day after extinction training. No significant differences were found between males and females. Ci) Averaged percent time spent freezing during each fear cue presentation during extinction training. Compared to the first trial of extinction, males showed significantly reduced freezing during trials 8–20. Freezing levels for females did not significantly decrease at any point in extinction training, with the exception of trials 14 and 19. #p<0.05, compared to trial 1. Cii) Averaged percent time spent freezing during each cue 1 day after extinction training. Males showed evidence of fear cue extinction retention. Females froze significantly more than males during the fear and fear+safety cues. Di) Averaged darting during each fear cue presentation during extinction training. No significant differences were found between males and females. Dii) Averaged darting during each cue 1 day after extinction training. Females had significantly higher dart levels than males during the fear cue, which was also significantly higher than the reward, safety and fear+safety cues in females. Means +/− SEM. #p<0.05, ##p<0.01 within sex, between cue/trial comparisons. *p<0.05, **p<0.01, ***p<0.001 within cue, between sex comparisons.

Figure 4.. No significant differences in shock reactivity between age-matched male and female rats.

A) Male and female rats (n=8 each) were subjected to increasing footshock intensities from 0.3mA to 1.0mA. No significant differences in freezing levels (means +/− SEM) were detected between males and females after each shock presentation. The box around the data at 0.5mA indicates the intensity used for the experiments in this study. There were no significant differences in the number of males or females who jumped (B) or darted (C) in response to the 0.5mA shock.