Experimental Design. A, Graphical representation of the three stages of the serial compound stimulus (SCS) conditioning paradigm and the context each stage took place in. Pre-Exposure to the SCS took place on Day 1 in Context A. Conditioning and Extinction took place in Context B. Animals only received footshock during Conditioning and were presented with shock-free SCS presentations during Extinction. B, Diagram illustrating the three SCS-shock association variants animals experienced during Conditioning. The Paired (PA) group experienced SCS co-terminating with shock, the Unpaired (UN) group experienced random presentations of the SCS and shock, and the Shock Only (SO) group experienced shock without SCS during Conditioning.

Statistical analysis of sex differences in behavior. To determine the extent to which sex influences behavior within each group, we conducted comparative analyses between males and females. The few significant sex differences we observed are detailed in the table. From left to right, the columns describe: Figure and/or panel that was analyzed for sex differences, the group that showed significant sex differences in behavior, the type of behavior analyzed, the statistical test performed, the p-value obtained, and the interpretation of the result. SCS, Serial compound stimulus; CD2, Conditioning Day 2; Ext2, Extinction Day 2; WN, white noise; PA, Paired; UN, Unpaired.

SCS-evoked freezing and activity during CD2 are affected by SCS-shock contingency. A, Trial-by-trial freezing to the tone period. B, Trial-by-trial freezing to the WN period. C, Trial-by-trial activity index to the tone period. D, Trial-by-trial activity index to the WN period. E, Comparison of average percentages of freezing to the tone period from all trials of CD2. F, Comparison of average activity index scores to the tone period from all trials of CD2. G, Comparison of average percentages of freezing to the WN period from all trials of CD2. H, Comparison of average activity index scores to the WN period from all trials of CD2. I, Comparison of baseline contextual freezing levels between all groups during CD2. J, Comparison of the difference in average freezing between pre-SCS and tone periods for the PA and UN groups from all trials of CD2. SCS, Serial compound stimulus; pre-SCS, 10 s prior to SCS; CD2, Conditioning Day 2; WN, white noise; PA, Paired; UN, Unpaired. Figures 2A-2D were analyzed using two-way ANOVA. Figures 2E-2H were analyzed using unpaired t-test with Welch’s correction. Figures 2I and 2J were analyzed using one-way ANOVA. Data from Figures 2A-2D and 2I are presented as Mean ± SEM. Data from Figures 2E-2H and 2J are presented as box-and-whisker plots from min to max. *p<0.05; ****p<0.0001.

SCS-shock contingency elicits jumping responses to white noise during CD2. A, Heatmap showing the percentage of the PA group that exhibited jumping to WN per trial on CD2. Jumping is distributed across 1 s bins, each coinciding with one of the ten pips of WN that occurred during each SCS presentation. B, Histogram detailing the cumulative distribution of jumps from 20 randomly selected subjects of the PA group across the duration of SCS from all 5 trials of CD2. Empty boxes represent each 0.5 s pip of tone, filled boxes represent each 0.5 s pip of WN, and striped boxes represent the 1 s shock stimulus. The vertical dotted lines depict the onset and termination of the WN period. Total jumps per stimulus are listed above histogram bars. C, Heatmap showing the percentage of the UN group that exhibited jumping responses to WN per trial on CD2. Jumping is distributed across 1 s bins, each coinciding with one of the ten pips of WN that occurred during each SCS presentation. D, Histogram detailing the cumulative distribution of jumps from the UN group across the duration of SCS from all 5 trials of CD2. Empty boxes represent each 0.5 s pip of tone, filled boxes represent each 0.5 s pip of WN, and striped boxes represent the 1 s shock stimulus. The vertical dotted lines depict the onset and termination of the WN period. ITI represents the period between SCS and shock. Total jumps per stimulus are listed above histogram bars. E, Dot plot showing the distribution of jumps across the duration of SCS from 20 randomly selected subjects of the PA group for each trial of CD2. Each dot represents a single jump event, and each tick on the x-axis represents the onset of each pip of tone or WN. The vertical dotted lines depict the onset of the WN period. F, Dot plot showing the distribution of jumps across the duration of SCS from the UN group for each trial of CD2. Each dot represents a single jump event, and each tick on the x-axis represents the onset of each pip of tone or WN. The vertical dotted lines depict the onset of the WN period. G, Survival graph showing when subjects of the PA and UN groups, represented as percentages of their respective cohorts, first exhibited jumping behavior to either portion of SCS. SCS, Serial compound stimulus; ITI, Inter-trial interval; CD2, Conditioning Day 2; WN, white noise; PA, Paired; UN, Unpaired.

SCS-shock contingency elicits darting responses to white noise during CD2. A, Heatmap showing the percentage of the PA group that exhibited darting responses to WN per trial on CD2. Darting is distributed across 1 s bins, each coinciding with one of the ten pips of WN that occurred during each SCS presentation. B, Histogram detailing the cumulative distribution of darts from 20 randomly selected subjects of the PA group across the duration of SCS from all 5 trials of CD2. Empty boxes represent each 0.5 s pip of tone, filled boxes represent each 0.5 s pip of WN, and striped boxes represent the 1 s shock stimulus. The vertical dotted lines depict the onset and termination of the WN period. Total darts per stimulus are listed above histogram bars. C, Heatmap showing the percentage of the UN group that exhibited darting responses to WN per trial on CD2. Darting is distributed across 1 s bins, each coinciding with one of the ten pips of WN that occurred during each SCS presentation. D, Histogram detailing the cumulative distribution of darts from the UN group across the duration of SCS from all 5 trials of CD2. Empty boxes represent each 0.5 s pip of tone, filled boxes represent each 0.5 s pip of WN, and striped boxes represent the 1 s shock stimulus. The vertical dotted lines depict the onset and termination of the WN period. ITI represents the period between SCS and shock. Total darts per stimulus are listed above histogram bars. E, Dot plot showing the distribution of darts across the duration of SCS from 20 randomly selected subjects of the PA group for each trial of CD2. Each dot represents a single dart event, and each tick on the x-axis represents the onset of each pip of tone or WN. The vertical dotted lines depict the onset of the WN period. F, Dot plot showing the distribution of darts across the duration of SCS from the UN group for each trial of CD2. Each dot represents a single dart event, and each tick on the x-axis represents the onset of each pip of tone or WN. The vertical dotted lines depict the onset of the WN period. G, Survival graph showing when subjects of the PA and UN groups, represented as percentages of their respective cohorts, first exhibited darting behavior to either portion of SCS. SCS, Serial compound stimulus; ITI, inter-trial interval; CD2, Conditioning Day 2; WN, white noise; PA, Paired; UN, Unpaired.

Representative comparison of PA and UN groups’ response to SCS during conditioning. Video 1 shows defensive responses to the last SCS during CD2 for the PA group and the UN group. The video features audio of the SCS, which consists of 10 pips of tone followed by 10 pips of WN. SCS, Serial compound stimulus; CD2, Conditioning Day 2; WN, white noise; PA, Paired; UN, Unpaired.

Tone-evoked freezing is associative and is reduced by extinction learning. A, Trial-by-trial percentage of freezing to the tone period for the PA, UN, and SO groups during Ext1 and Ext2. B, Comparison of the difference in average freezing to the tone period from early and late 4-trial bins of Ext1. C, Comparison of the difference in average freezing to the tone period of SCS from early and late 4-trial bins of Ext2. D, Comparison of the difference in average freezing between pre-SCS and tone periods during Ext1. E, Comparison of the difference in average freezing between pre-SCS and tone periods during Ext2. SCS, Serial compound stimulus; Ext1, Extinction Day 1; Ext2, Extinction Day 2; PA, Paired; UN, Unpaired; SO, Shock Only. Figure 5A was analyzed using two-way ANOVA. Figures 5B, 5C, 5D, and 5E were analyzed using one-way ANOVA. Data from Figure 5A are presented as Mean ± SEM. Data from Figures 5B, 5C, 5D, and 5E are presented as box-and-whisker plots from min to max. *p<0.05, ****p<0.0001.

Stimulus-induced flight is associative and is partially replaced by freezing during extinction. A, Trial-by-trial activity index to the WN period for the PA, UN, and SO groups during Ext1 and Ext2. B, Comparison of the difference in average activity index scores to the WN period from early and late 4-trial bins of Ext1. C, Comparison of the difference in average activity index scores to the WN period from early and late 4-trial bins of Ext2. D, Trial-by-trial percentage of freezing to the WN period of SCS for the PA, UN, and SO groups during Ext1 and Ext2. SCS, Serial compound stimulus; Ext1, Extinction Day 1; Ext2, Extinction Day 2; WN, white noise; PA, Paired; UN, Unpaired; SO, Shock Only. Figures 6A and 6D were analyzed using two-way ANOVA. Figures 6B and 6C were analyzed using one-way ANOVA. Data from Figures 6A and 6D are presented as Mean ± SEM. Data from Figures 6B and 6C are presented as box-and-whisker plots from min to max. **p<0.01.

Stimulus-evoked escape jumping and darting during extinction. A, Histograms detailing the cumulative distribution of jumps across the duration of SCS from the first 4 trials of Ext1 for 20 randomly selected subjects from the PA group, the entire UN group, and the entire SO group. Empty boxes represent each 0.5 s pip of tone, filled boxes represent each 0.5 s pip of WN, and the vertical dotted lines represent the onset and termination of the WN period. Total jumps per stimulus are listed above histogram bars. B, Histograms detailing the cumulative distribution of darts across the duration of SCS from the first 4 trials of Ext1 for 20 randomly selected subjects from the PA group, the entire UN group, and the entire SO group. Empty boxes represent each 0.5 s pip of tone, filled boxes represent each 0.5 s pip of WN, and the vertical dotted lines represent the onset and termination of the WN period. Total darts per stimulus are listed above histogram bars. C, Heatmap showing the percentage of each group that exhibited jumping responses to the WN period of SCS per trial on Ext1. D, Heatmap showing the percentage of each group that exhibited darting responses to the WN period of SCS per trial on Ext1. SCS, Serial compound stimulus; Ext1, Extinction Day 1; WN, white noise; PA, Paired; UN, Unpaired; SO, Shock Only.

Representative comparison of PA, UN, and SO groups’ response to SCS during an early and late extinction trial. Video 2 shows how defensive responses to SCS change from early extinction to late extinction during the first extinction session. The video features audio of the SCS, which consists of 10 pips of tone followed by 10 pips of WN. SCS, Serial compound stimulus; Ext1, Extinction Day 1; WN, white noise; PA, Paired; UN, Unpaired; SO, Shock Only.

Tail rattling is a non-associative behavioral response during extinction. A, Heatmap showing the percentage of each group that exhibited tail rattling behaviors to the tone period of SCS per trial on Ext1. B, Heatmap showing the percentage of each group that exhibited tail rattling behaviors to the WN period of SCS per trial on Ext1. C, Histogram detailing the cumulative amounts of tail rattles during the tone period of SCS across early and late 4-trial bins of Ext1 and Ext2. D, Histogram detailing the cumulative amounts of tail rattles during the WN period of SCS across early and late 4-trial bins of Ext1 and Ext2. SCS, Serial compound stimulus; Ext1, Extinction Day 1; Ext2, Extinction Day 2; WN, white noise; PA, Paired; UN, Unpaired; SO, Shock Only.