Network-Level Changes in the Brain Underlie Fear Memory Strength

Reviewer #1 (Public Review):

The authors demonstrate that reactivation of mild vs strong aversive contextual associations produces dissociable effects on fos expression across a wide network of relevant brain regions. Mild, 2-shock memory recruits a 'small-world' network in which amygdalar regions are functionally connected to other regions that modulate their activity and behavioral output, whereas strong, 10-shock memory isolates amygdalar nuclei from the rest of the network. These different patterns of correlated neural activity correspond with functional/behavioral differences - the authors confirm that weak, 2-shock memory is more effectively extinguished and is susceptible to reconsolidation relative to strong, 10-shock memory.

One major drawback of the manuscript is the fact that the data were collected from male subjects only. One might expect similar behavioral outcomes from male and female rats receiving 2-shock and 10-shock training. However, increasing attention to sex as a biological variable has revealed an interesting truth, namely that males and females can engage distinct neural pathways to arrive at the same behavioral destination. It should not be taken for granted that retrieval of aversive contextual associations would reproduce the same networks in females, and, as such, the manuscript does not give a complete accounting of the phenomenon under study.

The aversive associative memories described by the authors are characterized as mild or strong. More analysis of the meaning of memory strength, and its relationship to conditioning parameters, is needed. In particular, the authors should discuss issues such as amount of training, US magnitude, and rate of shock delivery. If amount of training is important, would 2 vs 10 presentations of a milder shock produce the same networks at retrieval? Would a larger shock require fewer presentations to isolate amygdalar regions from the rest of the network? If the shocks were presented at the same rate during training, would you get the same result in both groups? More data examining these questions would be ideal, but, in the absence of that, a discussion of these issues is needed and missing from the manuscript in its current form.

Reviewer #2 (Public Review):

The manuscript examined the behavioural and neural profile of weak and strong fear memories. The data provide strong evidence that weak but not strong fear memories are subject to extinction and reconsolidation disruption. Strong memories also show greater generalization. These differences were echoed in differential neural connectivity with weak fear memories showing greater connectivity between brains areas than strong fear memories.

Strengths:

The findings are of great importance and offer insight into why resistance to extinction and reconsolidation may underlie fear-related psychopathology.
The study uses key behavioural tests to study the durability of weak vs strong memories (extinction and reconsolidation) as well as studies the generalisation of those memories. These behavioural effects nicely dovetail with the neural connectivity analyses that were performed.

The data presented in this paper will be the basis for future hypothesis driven examinations on the causal influence of specific pathways involved in contextual fear.
Excellent use of the open field to control for motor effects.

Weaknesses:

One alternative account to the weak vs. strong memory distinction made in the paper is the opportunity for extinction in the 2S compared to the 10S group. In the 2S group, the last shock occurs in the 3rd minute, leaving 9 minutes of context exposure without reinforcement to follow. This is not the case for the 10S group. If context fear extinction is engaged during this time, then this would mean that two memories (acquisition and extinction) are taking place in the 2S group, weakening the fear memory in this group, setting up the ground for stronger effects of extinction, less generalization and of course potential greater connectivity required for representing and linking these memories. Indeed, the IL, a brain area linked to extinction, is more predominant in the connectivity map of the 2S compared to the 10S group. While testing this alternative is beyond the scope of this paper, it will need to be discussed.
Methodological detail is lacking re the timeline of study, and connectivity analyses.

Reviewer #3 (Public Review):

In this manuscript, Haubrich and Nader investigated the difference between mild and strong fear memory mechanisms at the circuit levels. Previous studies have shown the difference in mechanisms and functions of mild and strong fear memory. Interestingly, memory retrieval induces reconsolidation of mild fear memory, but not always strong fear memory; retrieved mild fear memory is disrupted by protein synthesis inhibition, whereas retrieved fear memory is more immune to this inhibition compared to mild memory. The authors measured c-fos expression following retrieval of mild and strong fear memories and compared functional connectivity of brain regions associated with retrieval of them using computation analyses. The authors suggested that mind and strong fear memories differ in neural networks at the circuit levels.
These are interesting findings.

Major concerns:

1. Previous studies including Karim's lab have shown that protein synthesis in the hippocampus is required for the reconsolidation of contextual fear memory and that the retrieval of contextual fear memory activates gene expression such as c-fos in the hippocampus. However, the authors failed to confirm this observation. This may be due to the small number of rats or some technical problems.

1. The author's computation analyses suggested differences in neural networks activated by the retrieval of mild and strong fear memories. The results of computer analysis are interesting. However, it is not clear whether such results are actually occurring in vivo. At this moment, the author's findings are not a conclusion, but rather a suggestion or hypothesis. Therefore, it is also important to conduct interventional experiments to evaluate the validity of the authors' findings. Specifically, the authors' results could be validated by analyzing the effects of inhibition of specific brain regions on mild and strong fear memories retrieval using such as DREADD and other methods. These experiments seem hard, but would greatly improve the quality of the manuscript.