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. 2024 Dec 16:12:RP90414.
doi: 10.7554/eLife.90414.

Contributions of associative and non-associative learning to the dynamics of defensive ethograms

Quan-Son Eric Le  1   2 , Daniel Hereford  1   2 , Chandrashekhar D Borkar  1   3 , Zach Aldaco  1   3 , Julia Klar  1   2 , Alexis Resendez  1   3 , Jonathan P Fadok  1   3
Affiliations

Contributions of associative and non-associative learning to the dynamics of defensive ethograms

Quan-Son Eric Le et al. Elife. .

Abstract

Defensive behavior changes based on threat intensity, proximity, and context of exposure, and learning about danger-predicting stimuli is critical for survival. However, most Pavlovian fear conditioning paradigms focus only on freezing behavior, obscuring the contributions of associative and non-associative mechanisms to dynamic defensive responses. To thoroughly investigate defensive ethograms, we subjected male and female adult C57BL/6 J mice to a Pavlovian conditioning paradigm that paired footshock with a serial compound stimulus (SCS) consisting of distinct tone and white noise (WN) stimulus periods. To investigate how associative and non-associative mechanisms affect defensive responses, we compared this paired SCS-footshock group with four control groups that were conditioned with either pseudorandom unpaired presentations of SCS and footshock, shock only, or reversed SCS presentations with inverted tone-WN order, with paired or unpaired presentations. On day 2 of conditioning, the paired group exhibited robust freezing during the tone period with switching to explosive jumping and darting behaviors during the WN period. Comparatively, the unpaired and both reverse SCS groups expressed less tone-induced freezing and rarely showed jumping or darting during WN. Following the second day of conditioning, we observed how defensive behavior changed over two extinction sessions. During extinction, the tone-induced freezing decreased in the paired group, and mice rapidly shifted from escape jumping during WN to a combination of freezing and darting. The unpaired, unpaired reverse, and shock-only groups displayed defensive tail rattling and darting during the SCS, with minimal freezing and jumping. Interestingly, the paired reverse group did not jump to WN, and tone-evoked freezing was resistant to extinction. These findings demonstrate that non-associative factors promote some defensive responsiveness, but associative factors are required for robust cue-induced freezing and high-intensity flight expression.

Keywords: associative learning; defensive behavior; fear; fear extinction; mouse; neuroscience; non-associative learning; pavlovian conditioning.

Plain language summary

Post-traumatic stress disorder (or PTSD for short) is a condition that can cause people to overreact to harmless cues, vividly re-experience a traumatic event, or freeze in place. To understand why this happens, researchers often study fear responses using an approach called fear conditioning, where laboratory animals learn to associate the sound of a tone with a mild electric shock. This conditioning causes animals to freeze with fear when they hear the tone. However, focusing on freezing overlooks the range of defensive actions animals may carry out, such as escaping or fighting. Capturing this complexity in experiments is important for understanding the dynamic nature of fear responses that occur in PTSD. Previous work showed that conditioning mice with a two-part cue, such as a tone followed by white noise, caused mice to freeze during the first cue and jump during the second cue. However, whether the mice learned this behaviour through conditioning or if it was an instinctive response to the cues remained unclear. To investigate this phenomenon, Le et al. – including some of the researchers involved in the previous work – conditioned mice with a variety of different cue combinations and monitored how they responded. As before, mice conditioned to associate a tone followed by white noise with an electric shock froze when they heard the tone and transitioned to jumping during the white noise. However, if during conditioning the sounds and shocks occurred at unpredictable times, the mice did not associate the sounds with the shock and therefore they froze less and rarely jumped. Similarly, reversing the order of the sounds so that the white noise happened before the tone also reduced jumping but not freezing. To investigate whether the mice could unlearn this fear response, Le et al. exposed the fear-conditioned mice to the cues without an accompanying electric shock. The mice that had been conditioned with a tone followed by white noise showed a weaker response to the cues, only freezing and not jumping. However, the mice with the reversed cues still froze even after this exposure, and the mice with the non-associated cues maintained very little freezing and jumping. Taken together, the findings suggest that while fear responses can be influenced by the association between certain noises and an electric shock, other factors such as the timing and the order of the sound cues can also impact the intensity of the fear response. The experiments also showed that this method of fear conditioning can be used for both learning and unlearning fear responses, revealing an approach for future studies into how fear responses change over time. Combining this more complex approach with other experimental techniques could help researchers identify the brain regions that drive fear responses, which may eventually benefit people with PTSD and other fear disorders.

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Conflict of interest statement

QL, DH, CB, ZA, JK, AR, JF No competing interests declared

Figures

Figure 1.
Figure 1.. Experimental design.
(A) Graphical representation of the three stages of the SCS conditioning paradigm. (B) Five SCS-shock association variants were used during conditioning. SCS, Serial compound stimulus; CD1, Conditioning Day 1; CD2, Conditioning Day 2; Ext1, Extinction Day 1; Ext2, Extinction Day 2; US, Unconditioned stimulus; ISI, Inter-stimulus interval.
Figure 2.
Figure 2.. Stimulus-evoked freezing and activity during CD2 are affected by serial compound stimulus (SCS)-shock contingency.
(A) Trial-by-trial freezing during the tone period. (B) Trial-by-trial freezing during the white noise (WN) period. (C) Trial-by-trial activity index during the tone period. (D) Trial-by-trial activity index during the WN period. (E) Average freezing during the tone period from all trials of CD2. (F) Average activity index scores during the tone period from all trials of CD2. (G) Average freezing during the WN period from all trials of CD2. (H) Average activity index scores during the WN period from all trials of CD2. (I) Baseline contextual freezing levels during CD2. (J) Differences in freezing between pre-SCS and tone periods from all trials of CD2. (K) Average activity index scores during the WN period for the PA and UN groups from all trials of CD2. (L) Average activity index scores during the WN for the PA and PA-R groups from all trials of CD2. PA: n=32, UN: n=20, PA-R: n=10, UN-R: n=10, SO: n=20. Data from (A–D) are presented as mean ± SEM and were analyzed with two-way ANOVA. Data from (E–J) are presented as box-and-whisker plots from min to max and were analyzed with one-way ANOVA and Tukey’s post-hoc multiple comparisons test. Data from (K and L) are presented as box-and-whisker plots from min to max and were analyzed with Welch’s unpaired t-test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ^p<0.05, effect of group. For both the standard and reverse SCS groups, (A, C, E and F) detail responses during the tone period, and (B, D, G and H) detail responses during the WN period.
Figure 3.
Figure 3.. Associative pairings of the serial compound stimulus (SCS) and shock lead to robust escape jumping during white noise (WN).
(A) The percentage of the paired (PA) group that exhibited jumping during WN on CD2. Data are distributed across 1 s bins, each coinciding with one of the ten pips of WN that occurred during each SCS presentation. (B) The cumulative distribution of jumps from 20 randomly selected subjects of the PA group across the duration of the SCS from all five 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 the histogram bars. (C) The percentage of the UN group that exhibited jumping during WN on CD2. Data are distributed across 1 s bins, each coinciding with one of the ten pips of WN that occurred during each SCS presentation. (D) The cumulative distribution of jumps from the UN group across the duration of the SCS from all five 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. ISI represents the period between SCS and shock. Total jumps per stimulus are listed above the histogram bars. € The distribution of jumps across the duration of the 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 line depicts the onset of the WN period. (F) The distribution of jumps across the duration of the 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 line depicts the onset of the WN period. (G) The percentage of the PA-R group that exhibited jumping during WN on CD2. Data are distributed across 1 s bins, each coinciding with one of the ten pips of WN that occurred during each SCS presentation. (H) The percentage of the UN-R group that exhibited jumping during WN on CD2. Data are distributed across 1 s bins, each coinciding with one of the ten pips of WN that occurred during each SCS presentation. (I) Total percentage of the cohort that jumped during WN over the whole CD2 session. (J) Total percentage of cohort that jumped to shock over the whole CD2 session.
Figure 4.
Figure 4.. Associative serial compound stimulus (SCS)-shock pairings elicit darting responses to white noise (WN) during CD2.
(A) The percentage of the paired (PA) group that exhibited darting responses to WN. Data are distributed across 1 s bins, each coinciding with one of the ten pips of WN that occurred during each SCS presentation. (B) The cumulative distribution of darts from 20 randomly selected subjects of the PA group across the duration of the SCS. 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 the histogram. (C) The percentage of the unpaired (UN) group that exhibited darting responses during WN. Data are distributed across 1 s bins, each coinciding with one of the ten pips of WN that occurred during each SCS presentation. (D) The cumulative distribution of darts from the UN group across the duration of SCS. 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. ISI represents the period between SCS and shock. Total darts per stimulus are listed above the histogram. (E) The distribution of darts across the duration of SCS from 20 randomly selected subjects of the PA group. 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) The distribution of darts across the duration of SCS from the UN group. 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) The total percentage of each group that jumped during WN over the whole session. (H) Average distance traveled during the preSCS period. (I) Average distance traveled during the tone period. (J), Average distance traveled during the WN period. (K) The total percentage of each group that jumped to shock over the whole session. PA: n=32, UN: n=20, PA-R: n=10, UN-R: n=10, SO: n=20. Data from (H-J) are presented as box-and-whisker plots from min to max and were analyzed with one-way ANOVA and Tukey’s post-hoc multiple comparisons test. *p<0.05; **p<0.01; ****p<0.0001.
Figure 5.
Figure 5.. Tone-evoked freezing in the paired (PA) group is reduced by extinction learning.
(A) Percent freezing during the tone period for the PA, unpaired (UN), and shock-only (SO) groups. (B) Percent freezing during the tone period for the paired reverse (PA-R) and unpaired reverse (UN-R). (C) The difference in average freezing during the tone period between the first and last four-trial bins of Ext1. (D) The difference in average freezing during the tone period between the first and last 4-trial bins of Ext2. (E) The difference in average freezing between pre-serial compound stimulus (SCS) and tone periods during Ext1. (F) The difference in average freezing between pre-SCS and tone periods during Ext2. PA: n=32, UN: n=20, PA-R: n=10, UN-R: n=10, SO: n=20. Data from (A and B) are presented as Mean ± SEM and were analyzed with two-way ANOVA and Tukey’s post-hoc multiple comparisons test. Data from (C–F) are presented as box-and-whisker plots from min to max and were analyzed with one-way ANOVA and Tukey’s post-hoc multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Figure 6.
Figure 6.. Stimulus-induced flight is associative and is partially replaced by freezing during extinction.
(A) Trial-by-trial activity during the white noise (WN) period for the paired (PA), unpaired (UN), and shock-only (SO) groups during Ext1 and Ext2. (B) Trial-by-trial activity during the WN period for the paired reverse (PA-R) and unpaired reverse (UN-R) groups during Ext1 and Ext2. (C) Difference in average speed during the WN period from the first and last four-trial bins of Ext1. (D) Difference in average speed during the WN period from the first and last four-trial bins of Ext2. (E) Trial-by-trial freezing during the WN period for all groups during Ext1 and Ext2. (F) Difference in freezing during the WN period from the first and last four-trial bins of Ext1. (G) Difference in freezing during the WN period from early and late four-trial bins of Ext2. PA: n=32, UN: n=20, PA-R: n=10, UN-R: n=10, SO: n=20. Data from (A, B and E) are presented as Mean ± SEM and were analyzed with two-way ANOVA and Tukey’s post-hoc multiple comparisons test. Data from (C, D, F and G) are presented as box-and-whisker plots from min to max and were analyzed with one-way ANOVA and Tukey’s post-hoc multiple comparisons test. #p=0.054, *p<0.05, **p<0.01, ***p<0.001.
Figure 7.
Figure 7.. Stimulus-evoked escape jumping and darting during extinction.
(A) The cumulative distribution of jumps from the first four trials of Ext1 for 20 randomly selected subjects from the paired (PA) group (top), the unpaired (UN) group (middle), and the shock-only (SO) group (bottom). Empty boxes represent each 0.5 s pip of tone, filled boxes represent each 0.5 s pip of white noise (WN), and the vertical dotted lines represent the onset and termination of the WN period. Total jumps per stimulus are listed above the histogram. (B) The cumulative distribution of darts from the first four trials of Ext1 for 20 randomly selected subjects from the PA group (top), the UN group (middle), and the SO group (bottom). 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 the histogram. (C) The percentage of each group that exhibited jumping responses during the WN period of serial compound stimulus (SCS) per trial on Ext1. (D) The percentage of each group that exhibited darting responses to the WN period of SCS per trial on Ext1. (E) Total percentage of each cohort that jumped to WN over the whole Ext1 session. (F) Total percentage of each cohort that darted to WN over the whole Ext1 session.
Figure 8.
Figure 8.. Tail rattling is a non-associative behavioral response during extinction.
(A) The percentage of each group that exhibited tail rattling to the tone. (B) The percentage of each group that exhibited tail rattling to the white noise (WN). (C) Cumulative tail rattling during tone across early and late periods of Ext1 and Ext2. (D) Cumulative tail rattling during WN across early and late periods of Ext1 and Ext2. (E) Total percentage of each cohort that tail rattled to tone during Ext1. (F) Total percentage of each cohort that tail rattled to WN during Ext1.
Author response image 1.
Author response image 1.. Baseline Freezing levels for all groups during the first extinction session.
Baseline period is defined as the first 180 seconds of the session, before any auditory stimulus was presented. PA, Paired; UN, Unpaired; SO, Shock Only; PA-R, Paired Reverse; UN-R, Unpaired Reverse. *p<0.05, **p<0.01, ****p<0.0001.
Author response image 2.
Author response image 2.. Trial-by-trial plot of activity index during the tone period of SCS across both extinction sessions for the PA group.
SCS, Serial compound stimulus; Ext, extinction; PA, Paired.
Author response image 3.
Author response image 3.. Trial-by-trial plot of activity index during the tone period of SCS across both extinction sessions for the SO group.
SCS, Serial compound stimulus; Ext, extinction; SO, Shock Only.
See this image and copyright information in PMC

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