Sexual differentiation of contextual fear responses

Abstract

Development and sex differentiation impart an organizational influence on the neuroanatomy and behavior of mammalian species. Prior studies suggest that brain regions associated with fear motivated defensive behavior undergo a protracted and sex-dependent development. Outside of adult animals, evidence for developmental sex differences in conditioned fear is sparse. Here, we examined in male and female Long-Evans rats how developmental age and sex affect the long-term retention and generalization of Pavlovian fear responses. Experiments 1 and 2 describe under increasing levels of aversive learning (three and five trials) the long-term retrieval of cued and context fear in preadolescent (P24 and P33), periadolescent (P37), and adult (P60 and P90) rats. Experiments 3 and 4 examined contextual processing under minimal aversive learning (1 trial) procedures in infant (P19, P21), preadolescent (P24), and adult (P60) rats. Here, we found that male and female rats display a divergent developmental trajectory in the expression of context-mediated freezing, such that context fear expression in males tends to increase toward adulthood, while females displayed an opposite pattern of decreasing context fear expression toward adulthood. Longer (14 d) retention intervals produced an overall heightened context fear expression relative to shorter (1 d) retention intervals an observation consistent with fear incubation. Male, but not Female rats showed increasing generalization of context fear across development. Collectively, these findings provide an initial demonstration that sexual differentiation of contextual fear conditioning emerges prior to puberty and follows a distinct developmental trajectory toward adulthood that strikingly parallels sex differences in the etiology and epidemiology of anxiety and trauma- and stressor-related disorders.

Figure 1.

Experimental design—experiments 1–4. (A) In experiment 1, subjects were conditioned with three tone–footshock pairings and then tested for retention, generalization and auditory fear either 1 or 14 d later. (B) In experiment 2, subjects were conditioned with five tone–footshock pairings and then tested for retention, generalization, and auditory fear 1 d later. (C) In experiment 3, subjects were placed into the conditioning chamber for either 5 or 15 min prior to receiving a footshock and then tested for retention 1 d later. (D) In experiment 4, subjects were exposed the context for either continuously for 15 min or spaced (1 min apart) across three, 5-min periods and returned to their homecages. The next day rats were returned to the same context and received an immediate footshock, 1 d later subjects were back for a retention test.

Figure 2.

Experiment 1: three trial delay conditioning: (A,B) post-shock freezing across three conditioning trials. (A) Female and (B) Male. (C,D) Three Trial Context Fear Memory—Context fear (context A) assessed either 1 (C) or 14 (D) d following the conditioning procedure. Total mean percentage of time freezing during a 4-min retention test plotted across developmental age in male and female subjects. (E,F) Three Shock Generalized Fear—context generalization (context B) measured at either 1 (E) or 14 (F) d following the conditioning procedure. Total mean percentage of time freezing during a 4-min generalization test. (G) Auditory fear test (context B). Total mean percentage of time freezing to three 30 sec tone alone presentations. (*) represents significant difference between age groups at the P < 0.05 criterion. (#) represents significant difference between sex P < 0.05 criterion.

Figure 3.

Experiment 2: five-trial delay conditioning: (A,B) post-shock freezing across trials in Females (A) and Males (B). Total mean percentage of time freezing assessed during the 1-min interval after each shock delivery. (C) Context fear (context A) assessed 1 d following the conditioning procedure. Total mean percentage of time freezing during a 4-min retention test. (D) Context generalization (context B) assessed 1 d following the conditioning procedure. Total mean percentage of time freezing during a 4-min generalization test. (E) Auditory fear test (context B). Total mean percentage of time freezing to five 30 sec tone alone presentations. (*) represents significant difference between age groups at the P < 0.05 criterion. (#) represents significant difference between sex P < 0.05 criterion.

Figure 4.

Experiment 3: One-trial context conditioning with 5 or 15 min placement-to-shock interval (PSI). Context fear (context A) assessed 1 d following the conditioning procedure. Total mean percentage of time freezing during a 4-min retention test comparing males and females. (A) 5 min PSI. (B) 15 min PSI. (*) represents significant difference between age groups at the P < 0.05 criterion (P19, P21, P24 significantly different than P60 in both males and females). (#) represents significant difference between sex P < 0.05 criterion.

Figure 5.

Experiment 4: context preexposure (CPE) facilitation procedures with spaced or continuous preexposure. Context fear (context A) assessed 1 d following the conditioning procedure. Total mean percentage of time freezing during a 4-min retention test comparing males and females. (A) Spaced CPE. (B) Continuous CPE. (*) represents significant difference between age groups (*) P < 0.05 and (***) P > 0.001 criterions. (#) represents significant difference between sex P < 0.05 criterion.