Sex Differences in Remote Contextual Fear Generalization in Mice

Abstract

The generalization of fear is adaptive in that it allows an animal to respond appropriately to novel threats that are not identical to previous experiences. In contrast, the overgeneralization of fear is maladaptive and is a hallmark of post-traumatic stress disorder (PTSD), a psychiatric illness that is characterized by chronic symptomatology and a higher incidence in women compared to men. Therefore, understanding the neural basis of fear generalization at remote time-points in female animals is of particular translational relevance. However, our understanding of the neurobiology of fear generalization is largely restricted to studies employing male mice and focusing on recent time-points (i.e., within 24-48 h following conditioning). To address these limitations, we examined how male and female mice generalize contextual fear at remote time intervals (i.e., 3 weeks after conditioning). In agreement with earlier studies of fear generalization at proximal time-points, we find that the test order of training and generalization contexts is a critical determinant of generalization and context discrimination, particularly for female mice. However, tactile elements that are present during fear conditioning are more salient for male mice. Our study highlights long-term sex differences in defensive behavior between male and female mice and may provide insight into sex differences in the processing and retrieval of remote fear memory observed in humans.

Keywords: contextual fear conditioning; fear generalization; fear memory; remote generalization; sex differences.

Figure 1

Schematic of contexts used in fear conditioning and generalization experiments. Context B was designed to be as perceptually distinct as possible from the training context (Context A). Context C retains the metal floor grid used in Context A, but is otherwise identical to Context B.

Figure 2

Remote contextual fear memory and generalization with perceptually distinct training and generalization contexts (Contexts A and B, respectively). (A) Experimental design and cohort information. (B) Effect of test order on freezing behavior in the training context (Context A) vs. a distinct novel context (Context B) at 3 weeks after standard contextual fear conditioning (CFC). Bonferroni post hoc comparisons following three-way analysis of variance (ANOVA) are indicated. (C) Discrimination index, calculated as % Freezing in Context A/(% Freezing in Context A + % Freezing in Context B). ^#p < 0.0001 for effect of test order in females, Bonferroni post hoc test following two-way ANOVA. *p < 0.05, ^#p < 0.0001. Error bars are mean ± standard error of the mean (SEM).

Figure 3

(A) Experimental design and cohort information. (B) Effect of fear conditioning protocol (cage-exposed mice with no shock vs. brief training protocol vs. standard CFC) in female mice (B→A test order) at 3 weeks after initial training. Significant Tukey post hoc comparisons following two-way ANOVA are indicated. ^#p < 0.0001. Error bars are mean ± SEM.

Figure 4

Contextual fear memory and generalization at proximal time-points (BA test order). (A) Experimental design and cohort information. (B) Comparison of freezing behavior in male and female mice at 24–48 h after conditioning, using distinct training and generalization contexts. Bonferroni post hoc comparisons following two-way ANOVA are indicated. (C) Discrimination index for males and females at 24–48 h after conditioning. *p < 0.05, **p < 0.01. Error bars are mean ± SEM.

Figure 5

Effect of context pre-exposure (0–2 sessions) on remote contextual fear generalization using distinct training and generalization contexts. (A) Experimental design. N = 30 mice per test order (AB or BA), with 15 mice for each pre-exposure condition. (B) Effect of test order on freezing behavior in the training context vs. novel context. Significant Bonferroni post hoc comparisons following two-way ANOVA are indicated. (C) Discrimination index calculations from previous data. Significant Bonferroni post hoc comparisons following one-way ANOVA are shown in graph. Dotted line indicates no-discrimination threshold. **p < 0.01, ***p < 0.001. Error bars are mean ± SEM.

Figure 6

Remote contextual fear generalization using context that retains metal grid floor used in the training context. (A) Experimental design. Context A and Context C are completely different in terms of odor, chamber shape, and lighting. However, the same metal grid floor through which shocks were delivered during training is present in both contexts. (B) Freezing behavior of male and female mice in remote contextual generalization, with both test orders (A→C and C→A). Significant Bonferroni post hoc effects following three-way ANOVA are indicated. (C) Discrimination index calculated from freezing data, with Bonferroni post hoc test following two-way ANOVA revealed a significant effect of test order in females. ***p < 0.001 and ^#p < 0.0001. Error bars are mean ± SEM.

Figure 7

Contextual fear memory and generalization at proximal time-points (C→A test order), in which the metal grid floor of the training context is retained in the otherwise contextually distinct generalization context. (A) Experimental design and cohort information. (B) Comparison of freezing behavior in male and female mice at 24–48 h after conditioning. (C) Discrimination index. Error bars are mean ± SEM.