Volume of Amygdala Subregions and Plasma Levels of Brain-Derived Neurotrophic Factor and Cortisol in Patients with s/s Genotype of Serotonin Transporter Gene Polymorphism of First-Episode and Drug-Naive Major Depressive Disorder: An Exploratory Study

Round 1

Reviewer 1 Report

The authors investigated association between amygdala volume, serotonin transporter gene polymorphism, catecholamine metabolite, BDNF and cortisol in first episode and drug naïve MDD. The authors could find several positive findings, that might shed light on pathophysiology of MDD. The manuscript is well written, and their conclusion is stated based on scientific results. I strongly suggest it should be accepted in the present form.

Author Response

Our reply

Thank you so much for your high evaluation of our manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

Despite many years of animal and human studies, Major Depressive Disorder (MDD) is one of the biggest challenges of modern society. Therefore, it's so important to improve knowledge regarding mechanisms and effective treatment of MDD. Unfortunately, despite the promising title, this manuscript does not bring scientists any foot forward in understanding MDD or any other type of depression.

 

The manuscript is poorly written, is rather speculative, and written in a non-scientific way. In my opinion that version is not ready to publish and every part needs to be almost completely rewritten. Below authors will find a list of major and minor issues which may help in case of resubmission.

 

MAJOR ISSUES:

 

1. inappropriate statistics. Actually, I really do not know how to comment on this issue since the authors seem to be aware that they use inadequate statistical analysis! We can read in chapter 4.6 “This was a preliminary and exploratory study; therefore, we used no correction for multiple testing”. Fact that the manuscript describes preliminary study DOES NOT ALLOW to use inappropriate analysis and make any conclusions based on this analysis. The authors appear to be deliberately deceiving the reader. 
2. Exaggerated conclusions from the results obtained, which in fact is an error of reasoning commonly known as "to infer the converse form a conditional". Based on presented measurements it can’t be concluded that: “Plasma levels of HVA were significantly negatively correlated with the left central nucleus and left paralaminar nucleus in the MD group. Plasma cortisol levels were significantly positively correlated with the left medial nucleus and right central nucleus in the MD group”. The authors don’t show ANY DATA which may support these conclusions. Correlation doesn't say anything about cause and effect.
3. I've been involved in neurochemical research for over 11 years, and it is a well-known fact that monoamines and their metabolites cannot cross the blood-brain barrier. Therefore IT CAN NOT BE CONCLUDED THAT the PLASMA LEVEL OF HVA OR ANY OTHER MONOAMINE AND ITS METABOLITE INFLUENCES BRAIN STRUCTURES. The authors will find this information in any pharmacology textbook.
4. Even in an abstract reality where monoamines and their metabolites can cross the blood-brain barrier this conclusion is absurd since there are no sufficient experiments and control groups in this manuscript to prove that statement. Is like saying that the Kyiv bombing increases the length of the day in Europe. Both take place but are not related to each other. Just because we observe A and B at the same time does not mean that A changes B or B changes A.
5. There is no logical explanation as to why the authors studied dopamine metabolites and cortisol in humans with polymorphism for the serotonin transporter.
6. A chaotic introduction that consists of statements loosely related to each other. Like:

• “On the other hand, there are reports suggesting an enlargement of the amygdala in patients with MD, which illustrate conflicting findings and the current knowledge gap in this field” - what is this gap?

• “Thus, s-allelic variants have less serotonin reuptake than l-allelic variants because of the reduced expression of 5-HTT mRNA [8][9]. This means that individuals with the s/s genotype are more sensitive to all stressful life events than those with the s/l or l/l genotype” - No it does not mean that! It's another statement which is "to infer the converse form a conditional" mistake. Less serotonin reuptake does not mean that someone is more sensitive to all stressful life events.

• “From the perspective of metabolites (...)” - metabolites can’t have perspective, they are not alive!

• “(...) however the amygdala subregion is not well studied because it is difficult to classify and identify the amygdala nucleus” - That’s not an argument! This sentence contradicts itself. If it were difficult to classify amygdala regions, we would not isolate them.
• “The relationships between plasma levels of catecholamine metabolites (HVA and MHPG), BDNF, cortisol, and amygdala subregions volume remains unknown.”  - So what? Many things remain unknown in neuroscience.

 

5. There are significant differences in gender proportion within experimental groups but authors claim that “the HC and MD groups were generally consistent.”
6. There is no explanation why left-handed participants were not excluded (as should be) from the study.
7. There is no need to describe insignificant results as an interesting trend. There is nothing wrong with reporting negative results. There is also no logical explanation why authors stress the trend in “the MD group tended to have a 103 lower estimated total intracranial volume than the HC group” where p = 0.18 and silence about HVA plasma level “differences” between groups where p = 0.13, or MHPG where p = 0.11.
8. Authors should provide U value in the text: “The Mann–Whitney U test showed no significant difference in the estimated total intracranial volume and plasma levels of substance between the HC and MD groups”
9. Authors have a tendency to overestimate their results like: “There was a significant difference in the effect on the left medial nucleus and plasma levels of cortisol in the HC and MD groups”. It is false. Authors do not make any intervention so they can’t write about effects. It’s only speculation.

MINOR ISSUES:

1. I’m guessing that authors use the term “Major Depression” instead of “Major Depression Disorder” included in DSM-V. If so, I strongly advise to use full and well-known terms.
2. The abbreviation for serotonin should be in line 50 not in 69.
3. Misspelling in the description of the figure. It should be “Anterior amygdaloid area” not “Anterior-amygdaloid-area-AAA”
4. Too sparse description of Figure 2A. It should look like in Figure 2B.
5. Inconsistent order of listed subregions of the amygdala.
6.  Lack of units for Estimated total intracranial volume in Table 2.

Author Response

Despite many years of animal and human studies, Major Depressive Disorder (MDD) is one of the biggest challenges in modern society. Therefore, it's so important to improve knowledge regarding the mechanisms and effective treatment of MDD. Unfortunately, despite the promising title, this manuscript does not bring scientists any foot forward in understanding MDD or any other type of depression. The manuscript is poorly written, rather speculative, and written in a non-scientific way. In my opinion this version is not ready for publication and every part needs to be almost completely rewritten. Below, authors will find a list of the major and minor issues that may help in case of resubmission.

 

Our reply

Thank you so much for your appropriate criticism. This will help improve our manuscript. Following your suggestions, we performed the appropriate statistical analyses and discarded the data of plasma catecholamine metabolites, which did not reflect what occurs in the brain. We answered all your comments in a point-by-point manner as follows.

 

 

Major issues

1 Inappropriate statistics. Actually, I really do not know how to comment on this issue since the authors seem to be aware that they used inadequate statistical analyses! We can read in chapter 4.6 “This was a preliminary and exploratory study; therefore, we used no correction for multiple testing”. The fact that the manuscript describes a preliminary study DOES NOT ALLOW for the use of inappropriate analyses and making any conclusions based on this analysis. The authors appear to be deliberately deceiving the reader.

 

Our reply

For the sections where exhaustive tests on the correlation between blood substances and amygdala subregions were carried out, we used the Benjamini–Hochberg procedure to control multiple comparisons.

 

2 There are exaggerated conclusions from the results obtained, which in fact is an error of reasoning commonly known as "to infer the converse form a conditional". Based on the presented measurements, it can’t be concluded that: “Plasma levels of HVA were significantly negatively correlated with the left central nucleus and left paralaminar nucleus in the MD group. Plasma cortisol levels were significantly positively correlated with the left medial nucleus and right central nucleus in the MD group”. The authors don’t show ANY DATA which may support these conclusions. Correlation doesn't say anything about cause and effect.

 

Our reply

Thank you. Based on your criticism, we deleted the results of the plasma levels of catecholamine metabolites, which did not reflect what occurs in the brain.

 

 

3 I've been involved in neurochemical research for over 11 years, and it is a well-known fact that monoamines and their metabolites cannot cross the blood-brain barrier. Therefore, IT CAN NOT BE CONCLUDED THAT the PLASMA LEVEL OF HVA OR ANY OTHER MONOAMINE AND ITS METABOLITE INFLUENCES BRAIN STRUCTURES. The authors will find this information in any pharmacology textbook.

 

Our reply

Thank you. We completely agree with your criticism. We withdrew the results of the plasma levels of catecholamine metabolites and the related discussion in the text.

 

4 Even in an abstract reality where monoamines and their metabolites can cross the blood-brain barrier, this conclusion is absurd since there are no sufficient experiments and control groups in this manuscript to prove that statement. It’s like saying that the Kyiv bombing increases the length of the day in Europe. Both take place but are not related to each other. Just because we observe A and B at the same time does not mean that A changes B or B changes A.

 

Our reply

Thank you for this comment. We completely agree with your criticism. We withdrew the results of the plasma levels of catecholamine metabolites and the related discussion in the text.

 

5 There is no logical explanation as to why the authors studied dopamine metabolites and cortisol in humans with polymorphism for the serotonin transporter.

 

Our reply

Thank you. We deleted the data of plasma catecholamine metabolites in the results. We focused on cortisol and serotonin transporter, and the following sentences were added to the text.

5-HTT knockout mutations have also been shown to moderate the adaptive response to early adverse environmental factors. The latter study used heterozygous 5-HTT knockout mice (having a 50% gene dose-dependent reduction of 5-HTT expression) and found that, although these mice did not show behavioral deficits when raised by mothers providing a lot of maternal care, they developed increased anxiety and depression-related behavior in adulthood when raised by mothers providing poor maternal care. It was proposed that such a gene-by-environment interaction could serve as a model for the increased vulnerability to early life stress in individuals with the 5-HTTLPR s-allele.

 

 

6 The introduction is chaotic and consists of statements loosely related to each other. Like:

 

“On the other hand, there are reports suggesting an enlargement of the amygdala in patients with MD, which illustrate conflicting findings and the current knowledge gap in this field” - what is this gap?

 

Our reply

Thank you for pointing this out. We revised the text as follows: On the other hand, some reports have suggested enlargement of the amygdala in patients with MD, which illustrates conflicting findings and inconsistency in the current knowledge in this field.

 

“Thus, s-allelic variants have less serotonin reuptake than l-allelic variants because of the reduced expression of 5-HTT mRNA [8][9]. This means that individuals with the s/s genotype are more sensitive to all stressful life events than those with the s/l or l/l genotype” – No, it does not mean that! It's another statement which is "to infer the converse form a conditional" mistake. Less serotonin reuptake does not mean that someone is more sensitive to all stressful life events.

 

Our reply

Thank you. We deleted the data of plasma catecholamine metabolites in the results. We focused on cortisol and serotonin transporter, and the following sentences were added to the text.

5-HTT knockout mutations have also been shown to moderate the adaptive response to early adverse environmental factors. The latter study used heterozygous 5-HTT knockout mice (having a 50% gene dose-dependent reduction of 5-HTT expression) and found that, although these mice did not show behavioral deficits when raised by mothers providing a lot of maternal care, they developed increased anxiety and depression-related behavior in adulthood when raised by mothers providing poor maternal care. It was proposed that such a gene-by-environment interaction could serve as a model for the increased vulnerability to early life stress in individuals with the 5-HTTLPR s-allele.

 

 

“From the perspective of metabolites (...)” - metabolites can’t have perspective, they are not alive!

 

Our reply

Thank you. We removed the relevant sections.

 

“(...) however the amygdala subregion is not well studied because it is difficult to classify and identify the amygdala nucleus” - That’s not an argument! This sentence contradicts itself. If it were difficult to classify amygdala regions, we would not isolate them.

 

Our reply

Thank you. We have deleted that section.

 

“The relationships between plasma levels of catecholamine metabolites (HVA and MHPG), BDNF, cortisol, and amygdala subregions volume remains unknown.”  - So what? Many things remain unknown in neuroscience.

 

Our reply

Thank you for this comment. We revised the text as follows:

Since the relationships between the amygdala subregion volume and the plasma levels of BDNF and cortisol have not been adequately studied, we aimed to elucidate this points in first-episode and drug-naive MDD patients with s/s genotype of 5-HTTLPR, who are vulnerable to stress. (Line136)

 

7 There are significant differences in gender proportion within experimental groups but authors claim that “the HC and MD groups were generally consistent.”

 

Our reply

We added the following text: “There were more females than males in the MD group.”

 

8 There is no explanation why left-handed participants were not excluded (as should be) from the study.

 

Our reply

Thank you for this comment. We added the following text:

In line with the study phase protocol, no exclusion of subjects was made based on the dominant arm.(Line 159)

 

9 There is no need to describe insignificant results as an interesting trend. There is nothing wrong with reporting negative results. There is also no logical explanation why authors stress the trend in “the MD group tended to have a lower estimated total intracranial volume than the HC group” where p = 0.18 and are silent about the HVA plasma level “differences” between groups where p = 0.13, or MHPG where p = 0.11.

 

Our reply

Thank you for pointing this out. We withdrew the data on the plasma levels of catecholamine metabolites from the text. We also deleted the corresponding sections.

 

10 Authors should provide the U values in the text: “The Mann–Whitney U test showed no significant difference in the estimated total intracranial volume and plasma levels of substance between the HC and MD groups”

 

Our reply

Thank you for this suggestion. As our statistical software could not calculate u-values, we have included the z-values, which are also statistical values.

 

11 The authors have a tendency of overestimating their results like, “There was a significant difference in the effect on the left medial nucleus and plasma levels of cortisol in the HC and MD groups”. It is false. The authors did not make any interventions so they can’t write about effects. It’s only speculation.

 

Our reply

Thank you for pointing this out. We deleted the part on interactions as it was pointed out that correlation does not imply causation.

 

Minor issues

1 I’m guessing that the authors used the term “Major Depression” instead of “Major Depression Disorder” included in the DSM-V. If so, I strongly advise that they use the full and well-known term.

 

Our reply

Thank you for this suggestion. We changed major depression (MD) to major depressive disorder (MDD).

 

2 The abbreviation for serotonin should be in line 50 not in 69.

 

Our reply

Thank you for pointing this out. We corrected the relevant section.

 

3 Misspelling in the description of the figure. It should be “Anterior amygdaloid area” not “Anterior-amygdaloid-area-AAA”

 

Our reply

Thank you for pointing this out. We removed the “AAA.” As for the order and hyphens, they were already ‘png data’ and could not be corrected.

 

4 The description of Figure 2A is too sparse. It should look like that of Figure 2B.

 

Our reply

Thank you for this comment. We added figure legends like figure 2A.

 

5 Inconsistent order of listed subregions of the amygdala.

 

Our reply

Thank you for noticing this. We re-laid out Table 2, Figure 2, Table 3, and Figure 3 so that they are in volume order.

 

6 Lack of units for Estimated total intracranial volume in Table 2.

 

Our reply

Thank you. We added mm³

Author Response File: Author Response.docx

Reviewer 3 Report

Structural changes in brain regions affected by mood disorders, such as the amygdala in the case of depression, present of course research interest, in order to complete missing knowledge of disease pathophysiology.  Still, there are several comments to be made, before considering publication of the manuscript, which you will find attached.

 

Comments for author File: Comments.docx

Author Response

Structural changes in brain regions affected by mood disorders, such as the amygdala in the case of depression, are of research interest. They could help fill the knowledge gap in disease pathophysiology. Still, there are several comments to be made before considering the publication of the manuscript. You will find these attached.

 

 

Our reply

Thank you so much for your comments. They helped us improve our manuscript. We answered all comments in a point-by-point manner as follows.

 

# Abstract

In the last sentence, the authors say that dopamine and cortisol may influence the amygdala regions as if a causal relationship has been found. This cannot be proved in a cross-sectional study, as the authors themselves correctly state in the discussion section. So, this sentence must be modified.

 

Our reply

Thank you for pointing this out. We revised the text as follows: No significant difference was observed in the amygdala total and subregion volumes between the HC and MD groups. No significant difference was found in plasma levels of BDNF and cortisol between the two groups. Also, no correlations were found between the total and subregion amygdala volume and plasma levels of cortisol or BDNF.

 

# Introduction

The introduction section seems somehow simplistic; it should be elaborate. For example:

 

1)           In paragraph 2, the authors state that individuals with the short allele (s/s) of the serotonin transporter have decreased serotonin reuptake compared with those with the long allele, hence are more vulnerable to stress. Why does this happen? The authors should more explicitly describe the link between the two.

 

Our reply

Thank you for this suggestion. We added more information on the relationship between the s/s gene and stress based on previous research.

 

 

2)           In paragraph 3, the authors should cite the literature referring to the possible interactions between cortisol, monoamines, and BDNF.

 

Our reply

Thank you for this suggestion. We added the following text: (Line127)

BDNF and cortisol have distinct roles in the physiology of the brain, but at the same time, there is an interaction between them. It is plausible that BDNF has an influence on the cortisol responsivity to stress, thereby implying a Met-allele in Val66Met polymorphism and cortisol integrative system. BDNF and cortisol undoubtedly play distinct and complementary roles in the physiology of the nervous system, in which cortisol proves to be the regulator of positive and negative effects. However, the detailed mechanism of the interaction between BDNF and cortisol for the pathophysiology of MD remains unknown.

 

3)           In paragraph 4, please substitute “nucleus” for “nuclei” (plural number)

 

Our reply

Thank you. We revised “nucleus” to “nuclei.”

 

4)           In the same paragraph, the authors should explain why investigating amygdala subregions is important. How is this going to help research? Are amygdala changes expected to be causes or consequences of depression pathophysiology?

 

Our reply

Thank you for this suggestion. In our study, there was a significant volume reduction in the lateral nucleus in patients with MD compared with that in HCs. The lateral nucleus of the amygdala is its main input structure and is required for all aspects of threat learning and associative plasticity. The structural changes in the lateral nucleus of the amygdala and anterior amygdaloid area might be related to the changes in neural circuitry, which might have resulted from the development and progression of diseases. The limbic-cortico-striatal-pallidal-thalamic (LCSPT) circuit plays an important role in understanding the neuroanatomical pathophysiology of depression.

 

5)           Finally, genes are written in italics, i.e., 5-HTTLPR instead of 5-HTTLPR.

 

Our reply

Thank you. We changed 5-HTTLPR to 5-HTTLPR.

 

# Results

Results are generally well presented, in a concise and comprehensive manner.

 

Our reply

Thank you so much for your high evaluation of our manuscript.

 

# Discussion

1)           The authors should try to explain more about the lateralization of the findings based on current knowledge. Also, please provide more information about the role of catecholamines, dopamine, and cortisol in depression.

 

Our reply

Thank you for your suggestions. We deleted the results of catecholamine metabolites, as they do not reflect what occurs in the brain according to reviewer#2. We focused on cortisol for the pathophysiology of depression.

 

We added the following sentences in the text to explain the role of cortisol and BDNF in depression;

(Line 258)

The hypothalamic–pituitary–adrenal (HPA) axis has been the focus of depression research. One of the most consistent biological findings in patients with severe MD with melancholic features, with dysregulation of the HPA axis, is the increased amount of plasma cortisol. This biological difference is due to a combination of excessive stress-related cortisol secretion and impaired glucocorticoid receptor-mediated HPA axis feedback inhibition. HPA axis changes are also associated with the impaired cognitive function of MD. Moreover, failure of HPA axis normalization with treatment is associated with poor clinical response and high relapse. Growth and adaptability at a neuronal level have been more broadly termed neuroplasticity, and it is possibly this neuroplasticity at a cellular level that is altered by inflammation and HPA axis dysfunction, both caused by environmental stress . The process of neurogenesis is controlled by nerve growth factors, including BDNF, which is reduced in patients with MD (PMID: 29387021: Kishi T et al, Front Psychiatry 2018), and reduced BDNF can be recovered with either pharmacotherapy or psychological interventions.

 

(Line286)

Diminished connectivity between the amygdala and the pregenual part of the anterior cingulate cortex (pgACC) during fear-related processing could be more vulnerable to anxiety as it pertains to greater circulating cortisol levels in daily life. In short, individual functional neural connective patterns of amygdala-hippocampal-pgACC circuit could play a role in the complicated link between cortisol and emotional related behaviors (PMID: 28814810, 2017, Hakamata). A recent report suggested that the basolateral amygdala to subgenual anterior cingulate cortex neural connectivity and cortisol-norepinephrine interaction, which may be associated with implicit memory bias, could be one of the pathophysiologies of anxiety disorders and MD (PMID: 34894424, 2022, Hakamata). BDNF is known to regulate synaptic plasticity and memory formation in many areas of the brain, including the amygdala, where BDNF signaling via tyrosine kinase B receptor (TrkB) is prominently involved in fear learning (PMID: 32845430, Meis, 2020). BDNF, acting through the TrkB, is thought to be a critical mediator of fear learning, and amygdala TrkB activation is required for the consolidation of stable extinction memories, which is involved in the pathophysiology of psychiatric diseases, including anxiety and mood disorders (PMID: 16783370). Pe-synaptic TrkB in BLA neurons has been reported to be necessary for memory extinction and contributes to BDNF signaling transduction from the basolateral amygdala to the infralimbic prefrontal cortex (PMID: 28749471, Li, 2017). In fact, MD patients showed impaired acquisition of conditioned fear (PMID: 33524676). The BDNF Val66Met polymorphism could be effects on amygdala-cortical connectivity during adolescence (PMID: 28387866). These findings suggest that the influence of cortisol and BDNF on the amygdala could play a role in the pathophysiology of MD. However, no significant correlations could be found in the amygdala volume and plasma cortisol of BDNF in the present explanatory research.

 

 

2)           Use of plasma instead of cerebrospinal fluid samples is an acceptable limitation, but the other two listed limitations are major, and apply to the methods section, as below:

#Methods

1)           Corrections for multiple comparisons should be done, sine qua non.

 

Our reply

Thank you. For the sections where exhaustive tests on the correlation between blood substances and amygdala subregions were carried out, we used the Benjamini–Hochberg procedure to control multiple comparisons.

 

2)           It is not clear to me why the authors have chosen to investigate only subjects who carry the s/s genotype. The authors should provide a sufficient explanation for the rationale of the research design.

 

Our reply

Thank you for this question and suggestion. We genotyped 49 HCs and 25 MD patients. Forty-six of the 49 HCs and all the 25 MD patients had s/s genotypes. In short, almost all participants had s/s genotype. Therefore, we excluded the three HC with the s/l genotype from the analysis.

 

3)           The authors state that the time for blood collection was not specific. This is very important because there is diurnal variation in cortisol secretion, which peaks early in the morning and diminishes during the rest of the day, depending on the time of sleep. BDNF, HVA, and MHPG may also exhibit diurnal variation. Therefore the time of collection of samples should be looked up for, if recorded, and this pitfall accordingly remediated, if possible; if not, I think that plasma measures may not be usable. 

 

Our reply

Thank you for this information. We precisely described the time for collection as follows in the text as your pointed out.

The participants fasted and rested for at least 30 min before blood sample collection. We performed blood sampling between 9 a.m. and 11 a.m. (Line 169)

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The manuscript has been mostly corrected. Some issues need to be still addressed:

line 57 - explain an abbreviation 5-HTT

line 61 & 84 - use shortcut 5-HT instead full name

line 80 - change "In short" to "For instance"

line 90 & 92 - provide references at the end of the statement

line 96 - change "adequately" to "sufficiently"

in lines 97 & 98 stays "who are vulnerable to stress" - explain how this vulnerability was assessed in the presented study

in line 178 stays "There were more females than males in the MDD group." while in the table is: "Sex, male/female" so it indicates that male is given before female and if so it was more males (!!) than females. Explain these discrepancies.

line 185 - explain abbreviations HVA and MHPG or delete

!! Table 2 there are the missing values for HVA and MHPG as suggested by the table description.

The order of listed subregions needs to be the same in Figures 1 & 2 as well as in table 3,  and the names of the subregions need to be corrected.

Figure 3 is actually a table, not a figure.

line 232, 299 & 301 - change "investigated" to "aimed to investigate"

line 281 - explain abbreviation BLA

There are missing abbreviations in the list of abbreviations at the end of the manuscript such as LCSPT, HVA, MHPG, pgACC, TrkB, and BLA.

 

 

Author Response

The manuscript has been mostly corrected. Some issues need to be still addressed:

>Thank you for your comments. We marked the amendments in purple.

line 57 - explain an abbreviation 5-HTT

> We changed Serotonin transporter (5-HTT) and added abbreviation.

line 61 & 84 - use shortcut 5-HT instead full name

>We changed serotonin to 5-HT

line 80 - change "In short" to "For instance"

> We change "In short" to "For instance"

line 90 & 92 - provide references at the end of the statement

>We added references at relevant (applicable) part. Reference number is 28, 29.

line 96 - change "adequately" to "sufficiently"

>We change "adequately" to "sufficiently"

in lines 97 & 98 stays "who are vulnerable to stress" - explain how this vulnerability was assessed in the presented study

>We deleted the phrase “Who are vulnerable to stress”.

in line 178 stays "There were more females than males in the MDD group." while in the table is: "Sex, male/female" so it indicates that male is given before female and if so it was more males (!!) than females. Explain these discrepancies.

>We changed as follow; "There were more males than females in the MDD group."

line 185 - explain abbreviations HVA and MHPG or delete!! Table 2 there are the missing values for HVA and MHPG as suggested by the table description.

>We deleted HVA and MHPG.

The order of listed subregions needs to be the same in Figures 1 & 2 as well as in table 3, and the names of the subregions need to be corrected.

>We corrected the figure design.

Figure 3 is actually a table, not a figure.

>We created it in Figure because on some websites (e.x. PubMed) coloring the Table is not reflected.

line 232, 299 & 301 - change "investigated" to "aimed to investigate"

>We change "investigated" to "aimed to investigate"

line 281 - explain abbreviation BLA. There are missing abbreviations in the list of abbreviations at the end of the manuscript such as LCSPT, HVA, MHPG, pgACC, TrkB, and BLA.

>HVA and MHPG were deleted. We added other abbreviations.

Author Response File: Author Response.docx

Reviewer 3 Report

Thank you for endorsing my comments. I think that the manuscript has substantially improved, and merits publication. Still, some correction of typos and minor spell check is needed

Author Response

Thank you for endorsing my comments. I think that the manuscript has substantially improved, and merits publication. Still, some correction of typos and minor spell check is needed.

 

>Thank you for your comments. We corrected whole text carefully.

Author Response File: Author Response.docx