Avalilação PREreview de Protein Synthesis Blockade Prevents Fear Memory Reactivation via Inhibition of Engram Synapse Strengthening

In the current work “Protein synthesis blockade prevents fear memory reactivation via inhibition of engram synapse strengthening”, the authors examine the role of proteostasis in memory retrieval following fear conditioning in mice, providing evidence that protein translation is necessary for both natural and optogenetically induced recall. Additionally, the authors demonstrate a strong association between translation dependent synaptic connectivity and memory recall suggesting a mechanism through which protein expression leads to increased synaptic connectivity between engram cells. The experiments provide compelling data that are generally in support of the conclusions drawn by the authors. In particular, the use of dual eGRASP is an elegant and direct way to assess changes in synaptic connectivity between multiple types of neurons, with data strongly supporting the conclusion that increased connectivity between engram cells (hippocampus) and basolateral amygdala is associated with increased memory recall, and depends on protein translation. However, there are a few areas that could help increase the overall clarity of the work.

Major Points

1.       The dual eGRASP and noncanonical amino acid (NCAA) labeling techniques were unclear. To help, the authors can include additional description of the methodologies in the main text which would tremendously increase the clarity of the work for the reader. The dual eGRASP, for example, could be expanded within the text to explain what the different colors represent and how to interpret the results for those less familiar with the technique especially given its importance to the main findings of the report. For the use of NCAA labeling, explaining how NCAA is being utilized to assess changes in nascent protein expression following CFC with or without protein translation inhibitors would be helpful.

2.       The conclusion that modification between engram cells is necessary for memory recall as presented here is somewhat correlative. In particular, no specific experiment was performed in which the authors directly block connectivity between engram cells and then assessed memory. Furthermore, the use of protein synthesis inhibitors to test this hypothesis, as stated in line 248, does not directly examine this hypothesis as loss of translation likely disrupts numerous cellular functions and not just connectivity. Disrupting connectivity between engram cells without indiscriminately ablating protein expression would more directly test this idea. Barring this experiment, the authors could include additional discussion around this point regarding other possibilities.

3.       The data provided clearly show increased structural connectivity between engram cells, and the use of Arc-Cre suggest increased activity between CA1 and BA. In addition to these studies, it would strengthen the claims that connections are functional synapses if the authors could provide additional assays to more clearly define changes specifically in synaptic transmission, such as calcium imaging or slice electrophysiology.

4.       In figure 3 C-E the SAL and 1XANI conditions show a small percent of freezing at baseline (1^st Off condition) while the 4XANI shows a near complete ablation of any freezing behavior. Could the apparent lack of freezing be due to hyperactivity induced by the loss protein translation rather than loss of recall? It would be helpful if the authors performed a locomotor analysis in the 4xANI vs SAL conditions to test this possibility, or if unable to then the authors could discuss this possibility in the text.

Minor Points

1.       It is unclear what the sample size for each experiment refers to. In the figure legends sample size is indicated as N = number, but what that number refers to (ie animals, images, cells, etc) is often ambiguous. Please provide what exactly the N refers to and if not referring to individual animals, it would be informative if you could also provide the in-mouse averages as well as how many animals were used.

2.       Clearer labeling of some images (e.g. Figures 1C, 2B, 4E, and S3C) to depict what each color represents would make it easier to understand for readers.

3.       In figure 2 the authors use noncanonical amino acid tagging to test various protein translation inhibitor applications following fear conditioning, claiming 4 injections of ANI showed comparable biotin labeling to animals that did not receive AHA. It would be helpful to provide a short explanation as to why this comparison was chosen in leu of the naïve or context only conditions.

4.       Many of the images and graphs are too compressed and thus grainy in appearance, making it difficult at times to properly evaluate their contents.

5.       In addition to these comments, an interesting future direction could be to identify any specific proteins that are increased that contribute to the increase in synapse density. The use of NCAA labeling would be an effective way to decipher nascent proteins involved with this process, and combing BONCAT mice with Arc-Cre would allow for increased specificity. Following biotin, the authors could probe for particular candidates including cell adhesion molecules known to be involved with synaptogenesis.

Competing interests

The author declares that they have no competing interests.

Use of Artificial Intelligence (AI)

The author declares that they did not use generative AI to come up with new ideas for their review.