Two separate groups of rats were submitted to the fear learning and fear
extinction paradigm described above, referred to as group 1 and group 2 (analyzed and discussed separately). High and low responder groups were determined by a median split of the average percent freezing of the last 10 minutes of day 3 cue-conditioned freezing& extinction learning, grouping rats into within session extinction (low responders) versus those who did not undergo within session extinction (high responders).
1.
Group 1 Behavior Results:
Although there were observed individual differences, all rats in group 1 acquired the conditioned fear on day 1 of the behavior paradigm (F[1,6]= 0.01; p=0.94) (Figure 3.1.a, e). Group 1 then shows high individual variation and overall poor context recall in both high and low responder groups, indicating poor fear memory recall (F[1,6]= 1.69; p=0.24) (Figure 3.1.b, e). Group 1 rats showed varied cue-conditioned freezing and extinction learning on day 3, but did not show significant differences between groups (F[1,6]=1.20; p=0.316) (Figure 3.1.c). Cued-fear recall and extinction learning, on day 3, can be broken into 2 different phases: the first few tone exposures (tones 1-5, minutes 5- 9) which indicate cue-fear recall in response to experiencing the CS in a new context, and the last 10 CS/tone presentations when the rats were undergoing within-session extinction
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acquisition, learning that the CS is no longer associated with the US. Group 1 shows varied cue-fear memory recall across individuals but did not show significant differences between groups (F[1,6]=0.60; p=0.698) (Figure 3.1.c, e). When examined alone, the last 10 tone presentations of cue exposure, indicating fear extinction learning, show
significant differences between high and low responders (F[1,6]=8.60; p=0.026) (Figure 3.1.c, e). Animals were grouped into high vs low responder groups based on the median split of freezing during this behavioral stage (median value = 21.77%). Group 1 rats showed individual variation during extinction recall on day 5, but did not show
significant differences between groups (F[1,6]=2.17; p=0.316) (Figure 3.1.d). Extinction recall, on day 5, can also be broken into 2 different phases: the first few tone exposures (tones 1-5, minutes 2-6) which indicate recall of the previously learned extinction memory, and the last 10 tone presentations indicating within session extinction learning for those individuals who had not yet undergone extinction learning, or extinction
learning reinforcement. Group 1, however, had very few individuals recall the previously acquired fear extinction memory and was not significantly different between high and low responder groups (F[1,6]=1.55; p=0.431) (Figure 3.1.d, e). Finally, both high and low responders undergo within session extinction learning (F[1,6]=2.75; p=0.148) (Figure 3.1.d, e).
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Figure 3.1: Group 1- Grouped Differences in Freezing Behavior During Fear and Extinction Paradigm. Group 1 rats were divided into high and low responder groups based on a median split of the mean percent freezing during the last 10 tones of day 3, c (median= 21.77%). There we no grouped statistical differences during any phase. Graph e breaks up the behavior paradigm into the learning segments outlined in Table 3.1 and shows that high and low responder groups show a statistical difference during day 3 cued fear extinction. Arows indicate tone presentation, lightninig bolts indicate shock presentation, asterisks indicate statistical significance (*=p<0.05).
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2.
Group 2 Behavior Results:
Although there were observed individual differences, all rats in group 2 acquired fear on day 1 of the behavior paradigm and did not show high vs low grouped differences (F[1,10]=2.46; p=0.148) (Figure 3.2.a, e). Group 2 then shows context recall and within session context extinction on day 2, which shows grouped differences between high and low responders, with high responders showing better context recall than the low
responders (F[1,10]=8.40; p=0.0159) (Figure 3.2.b, e). Grouped differences between high and low responders on day 3 cue-conditioned freezing and extinction learning was found to be significant (F[1,10]=14.47; p=0.0035)(Figure 3.2.c). Fear recall and extinction learning can be broken into 2 different phases: the first few tone exposures (tones 1-5, minutes 5-9) which indicate cue-conditioned freezing in response to experiencing the CS in a new context, and the last 10 CS/tone presentations when the rats were undergoing within-session extinction learning, learning that the CS is no longer associated with the US. All animals in group 2 showed good cue-condition freezing, indicating both high and low responders were able to recall the CS-associated fear memory (F[1,10]=4.61;
p=0.0572)(Figure 3.2.c, e). The last 10 tone presentations of cue exposure, indicating fear extinction learning, show stark differences in group 2 split between high and low responders, indicating low responders underwent cued fear extinction learning
(F[1,10]=17.58; p=0.0018) (Figure 3.2.c, e). Animals were grouped into high vs low responder groups based on the median split of freezing during this behavioral stage (median value = 34.27%).Grouped differences between high and low responders on day 5 extinction recall was found to be significant (F[1,10]=5.22; p=0.0454) (Figure 3.2.d). Extinction recall, on day 5, can also be broken into 2 different phases: the first few
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minutes of tone exposure (tones 1-5, minutes 2-6) which indicate recall of the previously learned extinction memory, and the last 10 tone presentations indicate within session extinction learning for those individuals who had not yet undergone extinction learning, or extinction learning reinforcement. Group 2 shows very clear distinctions between high and low responders extinction recall, where high responders demonstrated a recovery of the fear memory and subsequent freezing response and low responders demonstrated recall of the fear extinction memory (F[1,10]=13.23; p=0.0046) (Figure 3.2.d, e). Finally, both high and low responders undergo within session extinction learning (F[1,10]=0.32; p=0.585) (Figure 3.2.d, 3.2.e). Group 2 rats demonstrate clear grouped differences in the extinction processes, including acquisition and recall. This test does not indicate if recall differences are due to individual differences in ability to recall extinction memory or differences in ability to consolidate the extinction memory.
It is questionable as to if group 1 and group 2 rats can be combined. One reason is that that group 2 did not undergo ferret odor exposure prior to fear conditioning. Zoladz and Diamond’s 2016 review examined the effect predator-based psychological stress has on animal models used to study PTSD. The PTSD model examined equated predator exposure to “an inescapable, life-threatening experience”, a classified PTSD-inducing experiences. However, they found that predator exposure alone did not produce PTSD- like symptoms and that this may not produce symptoms translatable to humans (Zoladz and Diamond, 2016). Considering behavioral inconsistencies observed in group 1 and as of yet inconclusive results exploring the implications of ferret odor exposure on fear acquisition and fear extinction learning, group 1 was not included in imaging or correlation analysis.
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Figure 3.2: Group 2- Grouped Differences in Freezing Behavior During Fear and Extinction Paradigm. Group 2 rats were divided into high and low responder groups based on a median split of the mean percent freezing during the last 10 tone presentations of cued-fear recall & extincion acquisition, c (median= 34.27%). High vs low responder groups showed strong statistical differences during extinction acquisition (c, e) and extinction recall (d, e). Graph e breaks up the behavior paradigm into the learning segments outlined in Table 3.1. Arows indicate tone presentation, lightninig bolts indicate shock presentation, asterisks indicate statistical significance (*=p<0.05; **=p<0.01; ***=p<0.001).
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