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Sex differences in fear discrimination do not manifest as differences in conditioned inhibition

Distinguishing safety from danger is necessary for survival, but is aberrant in individuals with post-traumatic stress disorder (PTSD). While PTSD is more prevalent in women than men, research on sex differences in safety learning is limited. Here, female rats demonstrated greater fear discrimination than males in a CS+/CS− paradigm. To determine if this sex difference transferred to fear inhibition, rats were tested for conditioned inhibition in a summation test with the CS+ and CS− presented in compound; no sex difference emerged. The results suggest sex differences in the neural mechanisms of discrimination learning but not recall of a fear inhibitor.

Discrimination between safety and danger is necessary for survival. Incorrect evaluation of a stimulus as safe when it is dangerous could result in harm, while determining a stimulus as dangerous when it is safe results in unnecessary fear and anxiety. Further, when a safety cue is well learned, it can reduce fear in the presence of a danger cue, a learning phenomenon known as conditioned inhibition of fear (Kazama et al. 2013). Overgeneralization of fearrelated cues and aberrant conditioned inhibition of fear are seen in individuals with post-traumatic stress disorder (PTSD) (Jovanovic et al. 2012;Costanzo et al. 2016;Jenewein et al. 2016). Females are more likely to be diagnosed with PTSD than males (Kilpatrick et al. 2013;Kessler et al. 1995) and safety learning and discrimination are sensitive to hormonal birth control and trauma history in females compared with males (Gamwell et al. 2015;Lornsdorf et al. 2015). Translational research regarding sex differences in rodent fear discrimination is in its infancy, but has indicated greater discrimination in females compared with males, with later generalization of fear (Day et al. 2016). Biological sex is a significant factor in the expression of fear-based psychoses (Shansky 2015) and a better understanding basic behavioral differences in fear discrimination is needed to more fully realize the impact of sex for an individual's health (Shansky and Woolley 2016).
Intact male and normally cycling female adult Sprague-Dawley rats were used as follows: (n = 24/sex) for both fear discrimination acquisition and later fear discrimination recall, (n = 12/sex) for fear discrimination acquisition and conditioned inhibition, and an additional (n = 24/sex) that were given fear discrimination acquisition only, for a total N = 120. CS+/CS− discrimination conditioning adapted from Myers and Davis (2004) was used (Chen et al. 2016;. Conditioning trials began with a 5 sec, 1 kHz (75 dB) tone, followed by either the CS+ or CS− for 15 sec. CS+ trials coterminated with a 500 msec, 1.2 mA scrambled foot shock. Conditioning consisted of 15 presentations of each cue in quasi-random order, so that neither cue occurred more than twice in series with a 90 sec inter-trial interval. A flashing light or white noise pip were used as conditioning stimuli. Behavioral research has noted sex differences in fear expression, where many female rats exhibit an active responsetermed darting-when presented with fear stimuli, as opposed to the passive freezing response typically observed in males (Gruene et al. 2015a). A trained observer screened videos for evidence of darting, but in our experimental conditions darting occurred too infrequently to analyze. Therefore, freezing was used as a behavioral measurement of fear (Fanselow 1980) as previously (Christianson et al. 2011;Chen et al. 2016;. Freezing data were analyzed by analysis of variance (ANOVA) with sex as a between subjects factor and cue type and trial as within subjects factors with Sidak post hoc contrasts. Additional methodological details are provided in the online Supplemental Material.
Experiment 1 investigated sex differences in fear discrimination acquisition (n = 60/sex; Fig. 1) and recall (n = 24/sex; Fig. 2). Fear discrimination recall was tested the day after the initial conditioning session. The test began with 2 min of baseline context exposure, followed by 30 sec presentations of CS+ and CS− cues without presentation of shock. Each cue was presented 10 times in pseudorandom order with a 30-sec inter-trial-interval. Experiment 2 investigated sex differences in the acquisition and recall of conditioned inhibition (n = 12/sex; Fig. 3). Rats received five conditioning sessions as previously . Summation tests were similar to the recall test used in Experiment 1 with CS+/− trials added in which CS+ and CS− were presented in compound.
The tests began with 2 min of baseline context, followed by 30 sec of each cue-CS+, CS− and CS+/− -three times each in randomized order with a 30-sec inter-trial interval.
Females displayed discrimination earlier in conditioning than males (Fig. 1A). A three-way ANOVA of sex by cue by trial block (five blocks of three cue trials) revealed a main effect of trial block, F (4,472) = 40.579, P < 0.001, and interactions of trial by sex, F (4, 472) = 3.491, P = 0.008, and cue by trial, F (4,472) = 45.474, P < 0.001, but no significant interaction of trial block by cue by sex interaction, F (4,473) = 1.048, P = 0.382. Post hoc comparisons showed that females significantly discriminated between the CS+ and CS− in the first-trial block (P = 0.007), while males failed to show this discrimination until the second trial block (P = 0.769 on trial block 1; P = 0.004 on trial block 2). Females also displayed significantly reduced freezing to the CS+ compared with males on trial blocks 3, 4, and 5 (Ps = 0.039, 0.018, 0.003, respectively) as well as less freezing to the CS− compared with males on all trial blocks (P = 0.048 on the first trial block, Ps < 0.001 on trial blocks 2, 3, 4, and 5).
Freezing to each cue during conditioning is summarized in Figure 1B. ANOVA revealed main effects of sex, F (1,118) = 23.42, P < 0.0001, and cue, F (1,118) = 312.3, P < 0.0001, and a cue by sex interaction, F (1,118) = 22.15, P < 0.0001. Post hoc analyses showed that both sexes significantly discriminated between the CS+ and CS−, Ps < 0.0001, but that females displayed significantly less freezing to the CS−, P < 0.0001, compared with males. A discrimination index was calculated (freezing to CS+/freezing to CS− × 100) as a measure of animals' ability to discriminate between the CS+ and CS− (Fig. 1C). Males and females showed significantly different discrimination indices, t (118) = 6.343, P < 0.0001, with females showing more discrimination compared with males. The sex difference was consistent across all cohorts of animals (Supplemental Fig. 1).
Sex differences were evident in fear discrimination recall tests. An ANOVA of the CS trials across test and sex (Fig. 2B), revealed a main effect of trial, F (9,414) = 14.856, P < 0.001, and cue by trial interaction, F (9,414) = 3.333, P = 0.001, but no significant interactions of sex by trial, F (9,414) = 0.421, P = 0.924, or cue by trial by sex, F (9, 414) = 1.668, P = 0.095. Females significantly discriminated between the CS+ and CS− on the first trial (P < 0.001), and continued this level of discrimination throughout the test. Males also displayed immediate discrimination on trial 1 (P = 0.004) and match the discrimination level of the females throughout the remainder of the test (P < 0.001). While females' response to CS− was stable across trials, males reduced freezing on CS− presentations 7, 8, and 9 compared with trial 1 (Ps = 0.004, 0.003, and <0.001, respectively), as well as on trial 10 compared with trials 2 (P = 0.048) and 3 (P = 0.037), and on CS+ presentations 8, 9, and 10 compared with presentation 4 (Ps = 0.034, 0.03, and 0.007, respectively). Males and females significantly differed in their response to the CS+ on trials 1 (P = 0.019), 6 (P = 0.038), 7 (P = 0.019), 8 (P = 0.049), and 9 (P = 0.020) and showed even more differential responding to the CS−, with significantly different freezing on trials 1-6 and trial 8 (Ps < 0.032). Figure 2C shows average freezing to each cue during recall. ANOVA revealed main of effects of cue, F (2,92) = 93.01, P < 0.0001, sex, F (1,46) = 40.33, P < 0.0001, and cue by sex interaction, F (2,92) = 12.74, P < 0.0001. Post hoc analyses showed that females continued to freeze significantly less than males to the CS− (P = 0.0003), as well as to the baseline context at the start of the test (P < 0.0001). Females froze significantly less to context than all other cues (Ps < 0.01) and froze significantly less to the CS− than the CS+ (P < 0.0001). Males also froze significantly less to the CS− and baseline context compared with the CS+ (Ps < 0.0001), but freezing to the CS− and baseline context did not significantly differ (P = 0.641). This sex by cue interaction is summarized by significant difference in discrimination index (Fig. 2D, t (46) = 3.63, P = 0.0007). To indicate that the sex differences in cue response were not simply artifacts of differential baseline fear, we subtracted baseline freezing from average freezing to each cue. Here we find main effects of cue, F (1,46) = 248.6, P < 0.0001, sex, F(1,46) = 14.05, P = 0.0005, and a cue by sex interaction, F (1,46) = 8.692, P = 0.005. These differences indicate that the sex difference in discrimination is not only due to the sex difference in baseline fear, but that there is a general sex difference in discrimination between the CS and the conditioning context (Supplemental Fig. 2).
In Experiment 2 conditioned inhibition of fear was assessed in summation tests. On test 1, analysis on average freezing to each cue (Fig. 3B) in a 4 (cue) by 2 (sex) ANOVA revealed a significant effect of cue F (3,66) = 30.06, P < 0.0001, with significantly reduced freezing to CS− compared with the CS+ and CS+/− (Ps < 0.0001) and significantly increased freezing to the CS+ and CS +/− compared with baseline context (Ps < 0.0001). Animals did not discriminate between the CS+ and CS+/− in this test (P = 0.33). There was no main effect of sex, F (1,22) = 0.1599, P = 0.6936, or cue by sex interaction, F (3,66) = 0.8828, P = 0.4547. The difference in baseline context freezing that was observed in Experiment 1 was present in this smaller sample as a trend, but did not reach significance. This may reflect an effect of different estrous status on the test day between experiments, or sampling error and the intrinsic variability in this dependent measure, which is discussed in the online Supplemental Material. The lack of sex difference in simple CS+/CS− discrimination here is likely the result of differences in cue presentation in the summation test compared with the recall test. Specifically, the first presentation of the CS− in the summation test is as a compound with the CS+. In test 5 (Fig. 3C), the same analysis revealed a significant main effect of cue, F (3,66) = 35.72, P < 0.0001, and a significant cue by sex interaction, F (3,66) = 3.15, P = 0.0307, but no main effect of sex, F (1,22) = 0.1521, P = 0.7003. Post hoc analyses showed that animals displayed greater fear to the CS+ compared with the baseline context exposure, CS− and CS+/− compound (Ps < 0.0001). Animals also froze less to the CS− compared with the CS+/− and baseline context (Ps < 0.05).
We observed a marked difference in fear discrimination between male and female rats. Differential freezing to the fear CS+ and safe CS− was greater in females compared with males during the initial conditioning and in a recall test one day later, There were no sex differences in summation, but improved inhibitory summation on tests 4 and 5 compared with tests 1, 2, and 3 (Ps < 0.01). (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001.
Sex differences in fear discrimination www.learnmem.org furthermore, females exhibited greater initial discrimination between the context and the CS. These results are consistent with recent findings (Day et al. 2016), although in contrast to studies on fear discrimination in humans where females show less discrimination than males (Gamwell et al. 2015;Lornsdorf et al. 2015). Females also displayed lower contextual freezing, consistent with several prior reports in rodents (Pryce et al. 1999;Daviu et al. 2014;Pettersson et al. 2016, although see Keiser et al. 2017 for exception). When this difference in contextual freezing was subtracted from freezing to each cues, there was a larger difference in freezing to CS+, whereas the main difference in the uncorrected data was primarily in freezing to the CS−. This indicates that the sex difference may be in discrimination per se, rather than freezing to a particular cue. The discrimination test required that rats flexibily transition between fear and safe states, which may favor the inherently more active female rats (Gruene et al. 2015a,b). However, after several days of conditioning, females may accrue more fear to the CS− (Day et al. 2016) which could manifest as a transition from more active behavior early in conditioning to more passive, i.e., male-like, with additional conditioning and stress  and account for the lack of sex difference in conditioned inhibition summation tests. This outcome contrasts some of the results of Day et al. (2016) in which after repeated discrimination conditioning the CS− failed to pass a retardation test of conditioned inhibition. Whether these different empirical results are a consequence procedural differences or of different neural mechanism underlying summation and retardation phenomena remains unknown.
The sex difference in discrimination indicates that there may be sex differences in the neural circuitry underlying discrimination learning. Interactions between the amygdala and prefrontal cortex are critical for CS+/CS− discrimination (Likhtik et al. 2014) and sexual dimorphisms observed in humans include sex differences in amygdala anatomy (Ruigrok et al. 2014), amygdala response to negative or stressful emotions (Stevens and Hamann 2012;Kogler et al. 2015), and amygdala functional connectivity (Lopez-Larson et al. 2011;Engman et al. 2016). Sex differences have been found in the rodent medial prefrontal cortex (Baran et al. 2010;Fenton et al. 2014Fenton et al. , 2016, a brain region critical to fear expression, extinction, and discrimination (Sotres-Bayon and Quirk 2010; Milad et al. 2014;Sangha et al. 2014). Similarly, basolateral amygdala projecting neurons of the infralimbic region of the medial prefrontal cortex appear to differently mediate fear expression in males and females (Gruene et al. 2015b). That sex differences in safety learning do not persist in conditioned inhibition of fear also suggests that conditioned inhibition of fear occurs in a neural circuit that is distinguishable from the fear discrimination circuitry.