Auditory fear conditioning protocols and behavioral schedule

Auditory stimuli were paired with electrical shocks in both delay and trace fear conditioning (Fig. 1A). In delay fear conditioning (Fig. 1A1), the paired conditioned stimulus (CS+) was followed by an electrical shock at its termination, while the unpaired conditioned stimulus (CS-) was not. In trace fear conditioning (Fig. 1A2), the CS+ was followed by a shock after a 20-second trace interval. Mice underwent either delay or trace fear conditioning on Day 1, followed by re-exposure to the auditory stimuli during Extinction #1 on Day 2, Remote recall on Day 17, and Extinction #2 on Day 18 (Fig. 1B). After conditioning, mice were either allowed to sleep (S) or subjected to 6-hour SD.

Fig. 1

Protocols for delay and trace fear conditioning, followed by recall and extinction sessions. (A) In delay fear conditioning, an electrical foot shock was paired with the end of each CS+ (30 s, 10 kHz), while CS- (30 s, 2 kHz) was presented without a foot shock. In trace fear conditioning, the foot shock was delivered 20 s after the CS+ (30 s, 10 kHz). (B) Following auditory fear conditioning on Day 1, male and female mice were re-exposed to tone stimuli without the foot shock for Extinction #1 on Day 2, the Remote recall on Day 17, and Extinction #2 on Day 18. A subset of mice underwent 6-hour SD after auditory fear conditioning

Higher initial memory retrieval in delay fear conditioning compared to trace fear conditioning, and in females compared to males

We examined whether sex (male vs. female), test (delay vs. trace fear conditioning), or state (S vs. SD) influenced memory acquisition and initial recall. A three-way ANOVA (Table 1) on the last trials of Day 1 found no significant main effects for sex, test, or state, nor any interactions between these factors (Fig. 2A). However, during initial trials on Day 2, significant main effects were found for sex (F (1, 70) = 12.27, p = 0.0008) and test (F (1, 70) = 32.44, p < 0.0001), with no main effects of state or interactions (Fig. 2B). This indicates that females and mice in the delay conditioning exhibited higher freezing during initial fear memory recall, which was not affected by post-conditioning SD. We then examined the time course of freezing during recall and extinction sessions for each test and sex.

Table 1 Three-way ANOVA results for the last trials on day 1 and the initial trials on day 2Fig. 2

Higher freezing in initial recall in delay fear conditioning and in females. (A) During the last trials on Day 1, no significant differences were observed across test, sex, or state. (B) During the initial trials on Day 2, higher freezing levels were observed in the delay fear conditioning group and in females (**p < 0.01 for the main effect of the test factor, ##p < 0.01 for the main effect of the sex factor, three-way ANOVA, Table 1). Sample sizes for the delay fear conditioning group were n = 10 for male S, n = 10 for male SD, n = 9 for female S, and n = 10 for female SD mice. In the trace fear conditioning group, sample sizes were n = 10 for male S, n = 10 for male SD, n = 10 for female S, and n = 9 for female SD mice. Values represent the mean ± standard error of the mean (SEM)

Post-conditioning SD enhanced gradual fear extinction of delay fear memory

In males, both the S (n = 10) and SD (n = 10) groups exhibited increased freezing in response to both CS+ (Fig. 3A1) and CS- (Fig. 3A2) during delay fear conditioning on Day 1. During Extinction #1 on Day 2, both groups showed a significant decrease in freezing; however, only the S group restored high freezing levels during the Remote recall on Day 17, comparable to their initial recall on Day 2 (Fig. 3A1). Both male S and SD groups further reduced freezing to CS+ during Extinction #2 on Day 18 (Fig. 3A1). Additionally, low-level freezing was observed upon re-exposure to CS- on Day 2, 17, and 18 (Fig. 3A2), suggesting that SD did not affect motor functions or general fear or anxiety in male mice.

In females, both the S (n = 9) and SD (n = 10) groups showed increased freezing to CS+ (Fig. 3B1) and CS- (Fig. 3B2) during delay fear conditioning on Day 1. During Extinction #1 on Day 2, neither group showed significant decreases in freezing, with only mild reductions noted in the SD group (Fig. 3B1). In contrast, the SD group, but not the S group, exhibited significant reductions in freezing during post-extinction remote recall on Day 17 and in the early trials of Day 18. Unlike male S group (Fig. 3A1), the female S and SD groups did not show increased freezing from Day 2 to 17, potentially due to less effective extinction training with persistently high freezing on Day 2 (Fig. 3B1). As in males, low freezing responses to CS- were observed on Day 2, 17, and 18 in both female S and SD groups (Fig. 3B2).

A two-way ANOVA (Table 2) showed significant main effects of sex across early (Fig. 3C2, F (1, 35) = 11.61, p = 0.0017) and late (Fig. 3C3, F (1, 35) = 18.84, p = 0.0001) trials on Day 2, Day 17 (Fig. 3C4, F (1, 35) = 9.37, p = 0.0042), early (Fig. 3C5, F (1, 35) = 11.93, p = 0.0015) and late (Fig. 3C6, F (1, 35) = 6.36, p = 0.0164) trials on Day 18. Significant main effects of state were observed on Day 17 (Fig. 3C4, F (1, 35) = 8.56, p = 0.006) and early Day 18 (Fig. 3C5, F (1, 35) = 7.4, p = 0.0101). These results indicate that while sex influenced freezing during each set of trials for delay fear memory recall, post-conditioning SD reduced freezing during post-extinction remote recall in both males and females.

Table 2 Two-way ANOVA results for delay and trace fear conditioningFig. 3

In delay fear conditioning, post-conditioning SD enhanced gradual fear extinction. (A1) Male S mice (n = 10) showed decreased freezing during CS+ from Day 2, followed by Remote recall on Day 17 and re-extinction on Day 18. Male SD mice (n = 10) decreased freezing during CS+ on Day 2 without spontaneous recovery on Day 17 (*p < 0.05 and **p < 0.01 for S mice, #p < 0.05 and ##p < 0.01 for SD mice, multiple paired t-tests vs. initial 4 trials on Day 2). (A2) CS- triggered low-level freezing in male S and SD mice. (B1) Female S mice (n = 9) demonstrated decreased freezing during CS+ from Day 17. Female SD mice (n = 10) decreased freezing during CS+ from late trials on Day 2 without spontaneous recovery on Day 17. (*p < 0.05 and **p < 0.01 for S mice, #p < 0.05 and ##p < 0.01 for SD mice, multiple paired t-tests versus initial 4 trials on Day 2). (B2) CS- triggered low-level freezing in female S and SD mice. (C1) Similar freezing during CS+ was observed across sexes and states on Day 1. (C2 − 6) Female mice showed higher freezing during CS+ across early (C2) and late (C3) Day 2, Day 17 (C4), and early (C5) and late (C6) Day 18. SD reduced freezing during CS+ on Day 17 (C4) and early Day 18 (C5). (*p < 0.05 and **p < 0.01 for the main effect of the state factor, #p < 0.05 and ##p < 0.01 for the main effect of the sex factor, two-way ANOVA, Table 2). Values represent the mean ± SEM

Fig. 4

In trace fear conditioning, post-conditioning SD accelerated extinction. (A1) Male S mice (n = 10) showed mild decreases in freezing on Day 2 and strong decreases on Day 18. Male SD mice (n = 10) exhibited decreased freezing on Day 2 with further decreases on Day 18 (*p < 0.05 and **p < 0.01 for S mice, #p < 0.05 and ##p < 0.01 for SD mice, multiple paired t-tests vs. initial 4 trials on Day 2). (A2) Female S mice (n = 10) decreased freezing from Day 2 with further decreases on Day 18. Female SD mice (n = 9) showed strong decreases in freezing on Day 2 to levels comparable to Day 18. (*p < 0.05 and **p < 0.01 for S mice, #p < 0.05 and ##p < 0.01 for SD mice, multiple paired t-tests vs. initial 4 trials on Day 2). (B) SD decreased freezing on later Day 2 (B3) with no significant sex differences from Day 1 (B1), early (B2) and late (B3) Day 2, Day 17 (B4), to early (B5) and late (B6) Day18. (*p < 0.05 for state factor, two-way ANOVA, Table 2). Values represent the mean ± SEM

Post-conditioning SD accelerated trace fear extinction

Male S mice (n = 10) showed mild reductions in freezing on Day 2 and 17, with a more substantial decrease on Day 18 (Fig. 4A1). Male SD mice (n = 10), however, exhibited significant reductions on Day 2 and 17, with further reductions on Day 18. In females, while both S (n = 10) and SD (n = 9) mice began to decrease freezing on Day 2, with the SD group showing a more pronounced reduction (Fig. 4A2). A two-way ANOVA (Table 2) revealed significant main effects of state at late Day 2 (Fig. 4B3, F (1, 35) = 4.86, p = 0.0341), but no significant main effects of sex or interactions between state and sex. Thus, post-conditioning SD accelerated extinction of trace fear memory (Fig. 4B3) at an earlier stage than delay fear memory (Fig. 3C4, 5).

Controlling for inter-individual variability: ANCOVA results

Freezing during fear conditioning and recall is influenced by variability arising from individual differences in responsiveness to neutral and aversive stimuli, associative learning ability, defense strategies such as freeze or flight, among other factors. In females, this variability can also be affected by the estrous cycle. To account for these variations, we performed an analysis of covariance (ANCOVA), using freezing on Day 1 as a covariate, to isolate the effects of SD on recall and extinction while controlling for differences in memory acquisition.

In delay fear conditioning, ANCOVA (Table 3) revealed that SD significantly reduced freezing on Day 17 in male mice (Fig. 5A3, F (1, 17) = 8.12, p = 0.022), and on Day 17 (Fig. 5B3, F (1, 16) = 31.54, p = 0.004) and early Day 18 (Fig. 5B4, F (1, 16) = 18.71, p = 0.002) in females. In trace fear conditioning, SD significantly decreased freezing in females on late Day 2 (Fig. 6B2, F (1, 16) = 5.70, p = 0.021). The positive slope of all linear regression lines (Figs. 5 and 6) indicates that inter-individual variability was maintained across sessions. Thus, the enhancement of extinction in both delay and trace fear memory by post-conditioning SD is not due to differences in individual variability during memory acquisition.

Table 3 ANCOVA results for freezing behavior in recall sessionsFig. 5

Post-conditioning SD gradually enhanced delay fear extinction, independent of pre-existing group differences. (A, B) Scatter plots and linear regression lines representing freezing behavior in male (A) and female (B) mice during each set of CS+ recall trials compared to conditioning on Day 1. Group differences between S and SD were tested while accounting for freezing on Day 1 as a covariate (ANCOVA, *p < 0.05, **p < 0.01, Table 3). Each set of re-exposure corresponds to early Day 2 (A1, B1), late Day 2 (A2, B2), Day 17 (A3, B3), early Day 18 (A4, B4), and late Day 18 (A5, B5). SD decreased freezing during Remote recall in male mice (A3) and during Remote recall (B3) and early Extinction #2 (B4) in female mice

Fig. 6

Post-conditioning SD accelerated trace fear extinction, independent of pre-existing group differences. (A, B) Scatter plots and linear regression lines representing freezing behavior in male (A) and female (B) mice during each set of CS+ recall trials compared to conditioning on Day 1. Group differences between S and SD were tested while accounting for freezing on Day 1 as a covariate (ANCOVA, *p < 0.05, Table 3). Each set of recall trials corresponds to early Day 2 (A1, B1), late Day 2 (A2, B2), Day 17 (A3, B3), early Day 18 (A4, B4), and late Day 18 (A5, B5). SD decreased freezing during late Extinction #21 in female mice (B2)