The article by Ma, Zhang and colleagues observes the impact and involvement of BDNF signaling within the basal forebrain to reverse fear memory impairments induced by acute 6-hour sleep deprivation. It is an interesting study that incorporates behavioral (step-down inhibitory avoidance test) and neurochemical (western blotting and immunohistochemistry) assessments following bilateral microinjections of BDNF and ANA-12 (selective TrkB antagonist) to determine differences in sleep conditions. I find that the results on fear memory, sleep, and BDNF actions could be of great relevance to the literature following extensive revisions to the writing and requested clarifications. For future reviews, it would be of great help to incorporate line numbering by the text.

Please see my major and minor reviews/ notes below per section:

Abstract
1. For the introduction part, it would be helpful to include the background or reasoning behind the need to investigate this study objective. Is there a working hypothesis?
2. a. For the method part, 1st sentence, add “Adult” before male Wistar rats.
b. As BDNF has at least 2 receptors, such as TrkB and p75, it is important to already clarify that it’s the TrkB receptor.
c. Add “bilaterally” after “…ANA-12 were further injected…”

Introduction
3. In general, please note that when a word is first introduced, e.g. basal forebrain, to also put the abbreviation next to it (i.e. BF) if the abbreviation will be used in subsequent parts of the manuscript.
4. Second paragraph: to better introduce the BF, perhaps sentence could be rewritten as: Within the basal forebrain (BF), a region implicated in sleep-wake control, there are three major types of neurons: cholinergic, glutamatergic, and GABAergic neurons.
5. In the last sentence on page 4, you mention that the BF is involved in nearly all types of memories. It can be helpful for the reader to name 2 or 3 types of memories and add a few references.
6. My suggestion is to start the next paragraph with “Of interest, brain derived neurotrophic factor (BDNF)…”
7. At the end of the introduction, please clarify the hypothesis of the study.

Materials and methods
8. In section 2.2, please add the length of the sleep deprivation protocol.
9. Under Experiment 1, please revise sentence. See suggestion: “Immediately after sleep deprivation, fear training in the step-down inhibitory avoidance test was initiated.
10. The group breakdown is very confusing and would require better clarification from the sentence “Short term memory (STM)…” and onwards. For instance, you mention “another 16 rats”, so are the 16 rats from the groups that underwent STM and LTM assessments or are they the remaining animals from the RC and SD that didn’t undergo memory assessments since you had initially started with 18 animals per group, but only 10 per group were used to assess memory?
11. Under Experiment 2, name the 6 groups. They should be named in the text, and not only on the experimental timeline and figures.
12. It was not clear when the surgical procedure occurred. So here is my suggestion: “Animals underwent bilateral cannula implantation into the BF and were allowed 10 days of recovery post-operation”. This can be inserted before you mention the next sentence about sleep deprivation.
13. The last 2 sentences under Experiment 2 require restructuring and better flow. Furthermore, were the brains of the rats that underwent STM and LTM assessments subjected to Hematoxylin and Eosin staining for injection site verification?
14. In section 2.5:
a. Which rat brain atlas was used? Year?
b. What was the gauge of the dummy cannula and internal injector?
c. Did animals receive any post-operative drug immediately following the surgery? What was the concentration? Company?
15. In section 2.6:
a. Please explain a little bit more about ANA-12’s properties so that readers understand the reasoning behind the use of this particular antagonist in the context of your manuscript. One sentence is enough. It is possible to cite the original authors “Cazorla et al., 2011”.
b. Why was BDNF administered 10 minutes before training and ANA-12 administered 1 hour before training? Is this related to drug efficacy? Please explain.
c. Why were these dosages selected? References or previous laboratory pilot studies?
d. Which micro-infusion pump was used with the 10μl Hamilton Syringe? Which company?
16. In section 2.8:
a. Include RRID # for the BDNF antibody.
b. Which concentrations were used for the primary and secondary antibodies? Which company manufactured the secondary antibody?
c. How many times were sections washed?
d. Was there a blocking step prior to incubation with the primary antibody?
e. Was there Hoechst staining?
f. Were the cells counted or photomicrographs taken?
17. In section 2.9:
a. Please write out the concentrations of the antibodies used.
b. Write out the abbreviation for TBS-T along with the concentration of the Tween 20. For example: Tris-buffered saline with 0.1% Tween 20 (TBS-T).
18. In section 2.10, move the statistical software information to the 1st sentence.

Results
19. In general, I find that it is very important to include further parameters such as the f-values or effect sizes in the statistical reporting to allow for a more complete and useful interpretation of the statistical data because p-value only quantifies the probability that the result is due to chance.
20. In section 3.2, 1st sentence, correct BNDF to BDNF.
21. In section 3.5, please place add figure numbers, for example, at the end of the 2nd sentence, insert (Figures 6F-H).

Discussion
22. The discussion seems to be an integration of the effects without a well-rounded synthesis of the work. Furthermore, it left out any explanation of the ANA-12 results and its implications. Please address these findings.
23. Some paragraphs in the discussion belong to the introduction section, for example, last paragraph starting from page 15 to page 16 and parts of middle paragraph on page 17.
24. 3rd sentence could be rewritten as: “The results showed that bilateral microinjection of BDNF activated the BDNF-TrkB pathway and partly reversed short and long term impairments in fear memory induced by acute sleep deprivation”.
25. Next sentence should be removed. It seems randomly placed there: ANA-12 (Figure 4).
26. You report that ANA-12 reduced levels of p-TrkB and p-PLCγ1 and aggravated SD-induced fear memory deficits. It would be helpful for the reader to better understand why the ANA-12 microinjection exacerbated behavioral and neurochemical results. Could you provide a better explanation and an idea of the mechanisms involved? Is it brain region-specific?
27. Please clarify whether the article by Ventskovska et al., 2015 was referring to an acute or chronic sleep deprivation.
28. What are the implications for increased transcription of BDNF in modulating fear memory?

Figures and figure legends
29. Figure 2 legend: you wrote “cc, central canal”. Do you mean here the “corpus callosum” because the central canal may be the cerebrospinal fluid-filled space that runs along the spinal cord.
30. Figure 3 legend:
a. Sentence “(A and B) Fear memory...” could be rewritten as “STM and LTM fear memory was evaluated using a step-down apparatus, and were shown impaired by 6 h of sleep deprivation”.
b. What’s the magnification for the immunostaining image?
c. Typically, “Data are shown as mean ± SD” tend to be one of the last sentences in each legend.
31. Figure 5 and 6 legends: Is the result for animals that were tested in STM or LTM? Or animals that did not undergo any behavioral testing? If the latter, please state how long after the bilateral injections that the animals were euthanized.
32. Figure 1 (Experimental timeline):
a. Experiment 1, add “western blot and IHC” in the box for Long-term memory.
b. Experiment 2: Was HE staining only done for the rats subjected to the LTM?