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

Reward availability and the potential for danger or safety potently regulates emotion. Despite women being more likely than men to develop emotion dysregulation disorders, there are few studies investigating fear, safety and reward regulation in females. Here, we show that female Long Evans rats do not suppress conditioned freezing in the presence of a safety cue, nor do they extinguish their freezing response, whereas males do both. Females did show evidence of conditioned inhibition of darting, however. 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. In summary, females showed a significantly different behavioral profile than males in a task that tests 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.

this was only seen in approximately 10% of male rats tested (Gruene et al. 2015). In reward 86 studies that have included female rats, significant sex differences in response to drugs of abuse 87 have been seen, with female rats consistently self-administering drugs more rapidly than males 88 (Becker & Koob 2016). This seems at odds with the clinical evidence showing men typically 89 consume more drugs, such as alcohol, than women. However, data also indicate that alcohol   After reward pre-training, rats were then exposed to sessions consisting of reward, fear and 124 safety cues. The reward cue and sucrose reward were the same as the reward pre-training 125 sessions. The fear cue was paired with a 0.5mA footshock, and both the safety cue and 126 fear+safety cue did not result in footshock or sucrose.

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The percent time spent at or in the reward port during each cue across session was quantified 129 for each DC session (Figure  2b). Two-way repeated-measures ANOVAs showed a significant 130 cue by sex effect, as well as main effects of cue and sex for DC1 (Table  1)

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The percent time freezing during each cue across session was quantified for each DC session 141 ( Figure  2c). Two-way repeated-measures ANOVAs showed a significant cue by sex effect for 142 sessions DC2-4, as well as main effects of cue and sex for every session (Table  1)

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Females did not show a significant reduction for any session.

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The number of darts during each cue was also quantified for each DC session and expressed 152 as a dart rate (Figure  2d;; # darts/20s cue). Darting behavior during cue presentation was largely 153 absent until DC3 and DC4. Two-way repeated-measures ANOVAs showed a significant cue by 154 sex effect for DC4, as well as main effects of cue, for DC2-4, and sex, for DC1 and DC4 (Table   155 1). Post hoc Sidak's multiple comparisons test showed that, during DC4, females expressed 156 more darting behavior compared to males during both the fear cue (p<0.0001) and the 157 fear+safety cue (p=0.0079). The females also showed a significant reduction in darting behavior 158 during the fear+safety cue compared to the fear cue during DC4 (p=0.0166), suggesting some 159 level of conditioned inhibition present in the females, when darting is taken into account.

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Our experiments utilized a shock intensity of 0.5mA throughout this study. For this particular 225 intensity, we also noted the number of rats that jumped or darted in response to a 0.5mA shock.

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No sex differences in the number of rats jumping in response to the 0.5mA footshock were 227 observed ( 2: p>0.9). The number of female rats darting after the 0.5mA footshock was higher 228 than males, but not significantly ( 2: p =0.0769), with five of the eight female rats tested 229 exhibiting the behavior. A higher number of females darting in response to the footshock in this 230 test would still not explain the lack of conditioned inhibition of freezing in the females, as 231 freezing levels at 0.5mA was slightly lower than the males ( Figure  5A). Our results do not 232 definitively show, but do suggest, that females may respond more preferentially to a footshock 233 with a darting response.

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In this study, we show females exhibit a significantly different behavioral profile than males in a 236 task that tests for reward, fear and safety cue discrimination, as well as conditioned inhibition 237 and extinction. Female Long Evans rats showed more reward seeking early in training and 238 persistently high freezing levels to the fear cue when in the presence of a safety cue or after 239 fear extinction. When freezing data was re-assessed in the context of darting levels, females 240 categorized as 'darters' did show a significant reduction in freezing levels to the fear cue during 241 extinction, while 'non-darters' did not show any evidence of fear extinction. This data adds to the 242 growing body of evidence of sex differences in fear regulation and highlights the advantages of 243 using more complex learning paradigms with additional behavioral measurements.

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The buffering effects seen in these males were linked to prelimbic cortical neurons projecting to presentation has shown that brain regions dealing with approach and reward become activated 300 (Rogan et al. 2005). These findings have also been translated to using safety signals to 301 overcome anhedonia in rats (Pollak et al. 2008), showing that safety signals may also be regulating emotion in addition to conditioned behavior (Foilb & Christianson 2018). In our study, 303 females consistently showed elevated reward seeking behavior during the reward cue 304 compared to males beginning in the second reward pre-training session. This data appears 305 consistent with reward studies showing significant sex differences in response to drugs of 306 abuse, with female rats consistently self-administering drugs more rapidly than males (Becker & 307 Koob 2016). The increased reward seeking in females seen in our study remained until the end 308 of the first DC session at which point they were equivalent to the males. Interestingly, DC1 is the 309 first time the animals are exposed to footshock. Taking into account the lack of conditioned              Figure 3. Females do not show significant extinction of fear. A) Schematic depicting experimental outline. During extinction training both the reward and fear cues are presented in the same session without reinforcement. During the extinction test 1 day later all cues are presented without reinforcement. 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. Females spent significantly more time in the port than males during the safety cue. 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, 9 and 11-20. Freezing levels for females did not significantly decrease at any point in extinction training, with the exception of trial 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 post hoc differences found between males and females during extinction training. Dii) Averaged darting during each cue 1 day after extinction training. Females had a significantly higher dart rate than males during the fear cue. Means +/-SEM. #p<0.05, ####p<0.0001 within sex, between cue/trial comparisons. *p<0.05, **p<001, ****p<0.0001 within cue, between sex comparisons.  . Extinction differences in female darters versus non-darters. A) Darting behavior during the last discriminative conditioning session was used to split females into 'darters' and 'non-darters' to assess if extinction behavior is influenced. A median split of the number of darts during the fear cue during DC4 was used to categorize female rats as 'darters' and 'non-darters'. For 8 of the 10 females categorized as 'darters' during the fear cue, darting levels were also in the top 50% during the fear+safety cue. Bi, Bii) Averaged dart rates for each fear cue presentation during extinction training. No significant differences in dart rates were seen between groups during extinction training or test. #p<0.05, females had a higher dart rate during the fear cue compared to all other cues. Ci) Averaged percent time spent freezing during each fear cue presentation during extinction training. Compared to the first trial of extinction, females categorized as 'darters' showed a significant reduction in freezing levels during trials 6, 8-10, 14 and 17-20. Freezing levels for females categorized as 'non-darters' did not significantly decrease at any point in extinction training. #p<0.05, compared to trial 1. Cii) Averaged percent time spent freezing during each cue 1 day after extinction training. Darters exhibited less freezing to the fear cue than non-darters. *p<0.05. Means +/-SEM. shock intensity (mA) Figure 5. 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.