The WAG/Rij Rat Model of Depression Comorbid with Absence Epilepsy: Sex Differences and Neurochemical Mechanisms

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a well-designed and comprehensive study exploring sex differences in depression comorbid with absence epilepsy using the WAG/Rij rat model. The findings of this study are very straightforward. The authors use robust behavioral paradigms and neurochemical analyses to support the presence of sex-specific differences in depressive-like and anxiety-like phenotypes and corresponding monoaminergic alterations in key brain regions. The work is novel, methodologically solid, and potentially impactful for the fields of epilepsy, depression, neuropsychopharmacology, and sex-differences research. However, there are several areas needing clarification, improvement, and streamlining before it can be accepted for publication.

Major comments

1. Although it was discussed in the research limitation of this study, estrous cycle in female should be simply tested to give evidence that estrous cycle did not affect the tested results in this study. Females used in this study should be better excluded from the possible effects of estrous cycle because many female rats were employed in this study. This is a very core question in this study. Even a very simple test should be very helpful for validating the conclusion of this study.
2. Lines 331, 336, 348, 370, It seems that they are overinterpreted to treat trend level as significant p-values. Throughout the text such as p = 0.00, p = 0.1, “trends” seem to be treated as if meaningful.
3. The “Discussion” section about findings is at too length, sometimes without deeper interpretation. I recommend that several paragraphs could be shortened or merged to make it more concise because the core findings of this study are very straightforward. It is not necessary to provide so much long discussion.

Minor comments

1. Line 109, change “depressionlike” to “depression like”
2. Line 131, does this sentence describe male? But the “male” is missing.
3. Lines 396,397, “This implies that WAG/Rij rats, irrespective of sex, compared with Wistar rats, exhibit anhedonia, a key symptom of depression-like behaviour” can be revised to description like “These findings indicate that both male and female WAG/Rij rats exhibit anhedonia, an essential feature of depressive-like behavior, compared to Wistar controls.”

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

General Assessment

This manuscript investigates sex differences in depression-like behavior comorbid with absence epilepsy using the WAG/Rij rat model, with a comprehensive behavioral and neurochemical analysis. The study addresses an important gap by systematically comparing behavioral phenotypes between male and female animals and linking these phenotypes with monoaminergic alterations across multiple brain regions.

Overall, the study is well-designed, and provides a substantial amount of valuable data. The manuscript is generally clearly written, and the results are presented in a detailed and transparent manner. However, several conceptual, methodological, and interpretational issues should be addressed to strengthen the translational relevance and clarity of the work.

 

Major Comments

A significant flaw in this study is that there is no information provided for female rat estrous cycles. Ovarian hormones have been known to play an important role in either enhancing or suppressing behaviors, monoaminergic systems, anxiety, or depression-like symptom phenotypes. The authors were supposed to consider this point in their discussions.

Although the FST is widely used, its validity as a model for ‘depression-like behavior’ is already controversial. Climbing and swimming performance could ascertain changes in strategy or stress and motor reaction rather than indicate depression. The authors ought to be less dogmatic about their model and consider other explanations, especially concerning motor or stress reactions when epilepsy-related changes are involved.

The manuscript holds that WAG/Rij females specifically display the ‘anxious depression’ phenotype. Although the data clearly supports the existence of increased anxiety-like features within females, the point at which anxiety and depression can be differentiated is rather vaguely circumscribed. There is a clear requirement for a more integrated analysis regarding the particular measures that define anxiety versus depression within the phenotype.

While there is a great deal of data on monoamines, correlations between behavioral measures and neurochemical analyses are largely missing. Correlation analyses between signals like sucrose preference or immobility times and levels or ratios of monoamines should either be included or a discussion on missing correlations would make the paper more comprehensive.

The Discussion would be strengthened by a translational perspective. For instance: How do the clinically observed sex differences in dopaminergic and serotonergic systems in epilepsy patients relate to the clinical data? Do the findings suggest sex-specific therapeutic targets or treatment strategies?

Minor Comments

The authors alternate between “depression-like behavior,” “depressive pathology,” and “depressive phenotype.” More consistency in terminology usage is needed.

Though the data is mostly informative, some of the panels are too crowded with data. Resizing the data points or making the graphing easier (particularly for multi-panel graphics) would help with readability.

In some areas, p-values are presented in trends (for example, p = 0.06–0.09). It would be best if the authors clarified whether these are exploratory findings, without too greatly emphasizing them.

Conclusion

In my opinion, this study is a very valuable source of a comprehensive analysis of differences in depression-like and anxiety-like behaviors in combination with absence seizures in the WAG/Rij rat model of absence epilepsy. It is a great benefit to apply both behavioral and neurochemical strategies in one study. In order to make the paper even more valuable for understanding comorbidities of neuropsychiatric disorders in epilepsy models, all of these concerns, especially concerning the effects of the estrous cycle, considerations in behavioral tests, and their translational relevance, should be met in a revised version of the text. I suggest major revision of the manuscript.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript presents a novel investigation into sex differences in depression comorbid with absence epilepsy using the WAG/Rij rat model. It is among the first to comprehensively compare both behavioral and neurochemical profiles between male and female WAG/Rij rats and Wistar controls. Key strengths include a multi-test behavioral battery (light-dark choice, open field, forced swim, sucrose preference, splash test) and detailed neurochemical analysis across five key brain regions. The study addresses a clinically significant question regarding sex-specific comorbidities in epilepsy. However, several major methodological, analytical, and interpretational flaws significantly undermine the validity and impact of the conclusions. Major revisions are essential.

1. The figure uses '#' to indicate a trend difference (0.05 < p < 0.1), but it is not clearly explained in the figure legend, which may cause confusion for readers.
2. The article suggests that female WAG/Rij exhibit 'anxious-type depression,' but it does not cite clinical diagnostic criteria or biomarkers to support the validity of this subtype.
3. Discussion page 15: 'In other words, despite the similarity... remain in WAG/Rij rats.' The sentence structure is complex and can be broken down to improve readability.
4. The terms 'DAergic' and 'dopaminergic' are used interchangeably; and it is recommended to standardize the usage throughout the text to either 'DAergic' or 'dopaminergic'.
5. On page 22, line 16, Literature [6] is only the volume number is provided, without issue number or page numbers.
6. Methodological Clarification Regarding Estrous Cycle: The manuscript acknowledges the lack of estrous cycle monitoring as a limitation. This point should be strengthened in the Methods or Limitations section. Please provide a clearer rationale for not monitoring the cycle (e.g., potential for increased handling stress, as mentioned, or citation of studies showing minimal impact on the primary tests used). Alternatively, the authors should discuss how future studies could address this.
7. Streamlining of Results Presentation: The neurochemical results, while detailed, are text-heavy and can be challenging to follow. Consider using a graphical summary (e.g., a schematic figure) in addition to Table 1 to highlight the key regional and sex-specific changes in DA and 5-HT pathways. This would improve readability and impact.
8. Figure 5 (correlation scatter plots) is fragmented and inefficient. Combine the six panels into a single, well-labeled multi-panel figure for easier comparison. Ensure all axes are clearly labeled.
9. Table 1 is dense and difficult to read due to excessive spacing. Reformate to a more compact style while maintaining clarity. Consider moving detailed statistics (post-hoc p-values) to a supplementary table.
10. "WAG/Rij rats, regardless of sex, exhibited symptoms of depression-like behaviour." is too strong. State that both sexes showed some depressive-like behaviors.
11. Integration of Discussion on Neurochemical Mechanisms: While both DAergic and 5-HTergic dysfunctions are reported, their discussion remains somewhat separate. It is recommended to integrate these findings more explicitly in a dedicated subsection of the Discussion. Elaborate on how the interaction or combined deficit in these two monoaminergic systems might specifically underlie the “anxious depression” phenotype observed in females, rather than treating them as parallel findings.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors addressed all concerns! I recommend that it can be accepted for publication. Congratulations!