Chloride Homeostasis Failure in Human Disease: KCC2/NKCC1 Microdomain Dysfunction as a Driver of Cortical Network Collapse

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript addresses an interesting and understudied topic in the field of neuroscience: alterations in chloride ion homeostasis (KCC2/NKCC1 microdomain) in human diseases. Undoubtedly, the work adequately summarizes current knowledge on the subject and clearly demonstrates the need to explore the field of ionic physiology to contribute to the understanding of neuronal function and dysfunction. Overall, the manuscript is well-structured and written; it allows for a smooth reading experience and is supported by robust evidence. The conclusions are appropriate to the study's objective and the scientific arguments presented. Regarding the cited literature, fundamental studies and recognized reviews in the field are mentioned; approximately three-quarters of the references are up-to-date. A few specific observations are made about the work:

-In the statement, “Under certain conditions glutamatergic signaling may also regulate the orientation of helices that determine the geometry of the ion-binding cavities,” what are those specific conditions?

-In the statement, “Also, certain glial populations exhibit persistent NKCC1 expression beyond the time of the transition of neurons, providing an additional layer of ionic regulation to the extracellular environment, and supporting network oscillations that rely on chloride-mediated mechanisms [37],” what are these glial populations?

-What do the authors consider to be some limitations of their manuscript proposal?

-Line 184, a comma is needed before the word “including.”

-Line 190, a comma is needed after the word “conditions.”

-Lines 210, 239, 248…, why is the word microdomain sometimes separated by a hyphen and sometimes not? The spelling of words used throughout the work should be consistent; for example, microdomain/micro-domain, microcircuits/micro-circuits, GABAA/GABA(A).

-Lines 213 and 214: Commas are missing after the words “dendrites,” “organization,” and “inhibitory.”

-In Figure 1, the chloride ion is missing its negative charge. The authors are advised to make the images slightly larger.

-Line 299: A comma is missing after the word “stability.”

-Lines 331 and 332: Correct to pathological stress.

-Line 373: Correct to “produce.”

-Line 441: Should read reactivation.

-Line 790: The word postmortem should be italicized due to its Latin roots.

Comments on the Quality of English Language

A thorough review of the manuscript is recommended. Some points to address are mentioned in the comments section for authors.

Author Response

Dear Esteemed Academic Reviewer,

We would like to thank you for your careful evaluation of our manuscript and for your thoughtful and constructive comments. We are especially grateful for your positive assessment of the structure, clarity, and scientific relevance of the work, as well as for recognizing the importance of investigating chloride homeostasis as a fundamental aspect of neuronal function and dysfunction. Your feedback was both encouraging and highly valuable in further refining the manuscript.

In addition to addressing your specific comments below, we would like to note that, following the recommendations of another reviewer, we undertook a comprehensive revision of the manuscript to improve clarity, reduce unnecessary descriptive detail, and strengthen conceptual focus. This included a substantial condensation of the Abstract and Sections 1–7, removal of metaphorical language, improved schematic presentation of Sections 2 and 3, redesign of Table 1 into a more compact and conceptual format, and refinement of the overall academic tone. We believe these global revisions have significantly enhanced the readability and coherence of the manuscript.

We provide below a point-by-point response to your comments.

Comment 1

“Under certain conditions glutamatergic signaling may also regulate the orientation of helices that determine the geometry of the ion-binding cavities,” what are those specific conditions?

Response 1

Thank you very much for this important request for clarification. We have revised the sentence accordingly to make these mechanisms more explicit and to avoid ambiguity.

Comment 2

“Also, certain glial populations exhibit persistent NKCC1 expression…” what are these glial populations?

Response 2

We are grateful for this request for clarification.

Comment 3

“What do the authors consider to be some limitations of their manuscript proposal?”

Response 3

Thank you for this thoughtful and important question. In response, we have added a dedicated statement in the revised manuscript addressing the limitations of our review.

Comment 4 (Language and punctuation corrections)

We are very grateful for these careful observations. All suggested corrections have been implemented in the revised manuscript. We greatly appreciate your attention to these details.

Comment 5

“Spelling of words should be consistent (microdomain/micro-domain, etc.)”

Response 5

Thank you for highlighting this important issue. We have carefully revised the manuscript to ensure full consistency in terminology.

Comment 6

“In Figure 1, the chloride ion is missing its negative charge. The authors are advised to make the images slightly larger.”

Response 6

We thank you for this valuable observation. The figure has been revised to correctly indicate the negative charge of the chloride ion (Cl⁻). In addition, we have increased the size and clarity of the figure to improve readability and visual presentation.

 

Once again, we would like to express our appreciation for your thoughtful review, your constructive suggestions, and your encouraging evaluation of our work. Your comments have helped us improve both the clarity and rigor of the manuscript.

We hope that the revisions we have made adequately address your concerns and further strengthen the manuscript.

With profound appreciation!

Reviewer 2 Report

Comments and Suggestions for Authors

In the review"Chloride Homeostasis Failure in Human Disease: 2 KCC2/NKCC1 Microdomain Dysfunction as a Driver of Cortical Network Collapse", the authors presented a review of the current literature with an attempt to conceptualize the aspect of the inhibitory function of chloride microdomains with a view to further development as a target for translational therapy. The review is of great reader interest. The following comments arose during the review of the article.

1. The Abstract section is unnecessarily verbose. The authors should choose the main directions of the review and/or the main idea of the review with a brief justification of the main conclusions of the work.
2. In section 1, the authors tell an interesting story about chloride homeostasis, but it is recommended to use a more academic language, without metaphors "the chloride ion is regulated as a mobile entity" p. 74.
3. It is recommended to shorten the descriptive part of section 1 somewhat, making the content of this section more concentrated and informative.
4. The authors illustrate section 2 well with a diagram. However, in the text of the review, they mention that "High-resolution optical and electron microscopy have shown a high density of KCC2 distribution in nanometer domains" pp. 210-211. It would be very clear if the authors illustrated these data in the form of micrographs, with reference to the original sources.
5. In section 2.1, the authors provide many descriptive characteristics. This section should be made more schematic and shortened.
6. Section 2.3 should also be shortened by removing the redundant description. In particular, the information on pages 290-299, as well as 299-306, can be significantly reduced if the authors highlight the main ideas in this section. Due to the abundance of details, the authors do not always manage to convey the main idea to the readers.
7. The authors' conclusion to section 2.3, pp. 317-325, should be made clearer and more readable.
8. In section 3.1, the authors should shorten the description on pages 335-343 and 344-353. Authors should not go into too much detail, artificially increasing the volume of the article; on the contrary, they should strive to find generalizations and present the material more concisely and constructively.
9. In section 3.2, the authors should shorten the description on pages 409-418. This and other sections of the review (see above) should be critically reviewed by the authors for generalization and reduction of details unrelated to the main idea of the section.
10. The authors presented a large amount of material in Table 1. It is necessary to title this table more clearly and less verbosely.
11. The authors should review the content of section 5 according to the above scheme (reduce redundant descriptions, make necessary improvements, and possibly provide specific examples).
12. Sections 6 and 7, which provide and discuss specific pathological conditions of humans with impaired chloride metabolism, as well as therapeutic frontiers through microdomain engineering, are very useful and informative. The authors should carefully review and edit these sections again, reducing unnecessary descriptive details.

Comments on the Quality of English Language

English needs editing

Author Response

Dear Esteemed Academic Reviewer,

We would like to express our gratitude for your careful reading of our manuscript and for the thoughtful, constructive, and insightful comments you provided. We greatly appreciate your recognition of the potential interest and relevance of this review, as well as your specific recommendations for improving its clarity, structure, and readability. Your observations were extremely valuable and helped us substantially refine the manuscript.

In response to your comments, we carefully revised the text throughout the manuscript with particular attention to reducing unnecessary descriptive detail, improving conceptual clarity, strengthening academic tone, and highlighting the principal ideas more directly and concisely. We also revised the abstract, retitled and redesigned Table 1 to make it more compact and informative, and substantially reworked several sections to improve readability and schematic presentation while preserving the scientific depth and mechanistic rigor of the review.

Below, we provide a detailed point-by-point response to each of your comments.

Comment 1

“The Abstract section is unnecessarily verbose. The authors should choose the main directions of the review and/or the main idea of the review with a brief justification of the main conclusions of the work.”

Response 1

Thank you for this important observation. We fully agreed that the original abstract was overly expansive and did not present the central directions of the review with sufficient economy and focus. In response, we substantially revised the abstract to make it more compact, denser, and more directly centered on the core conceptual message of the manuscript. The revised version now emphasizes the principal thesis of the review—namely, that chloride microdomains represent a spatially organized determinant of inhibitory polarity and cortical stability—while more briefly summarizing the molecular, spatial, and circuit-level mechanisms discussed in the article, as well as the main translational implications. We believe that the new abstract is significantly clearer, more informative, and more consistent with the reviewer’s recommendation.

Comment 2

“In section 1, the authors tell an interesting story about chloride homeostasis, but it is recommended to use a more academic language, without metaphors ‘the chloride ion is regulated as a mobile entity’ p. 74.”

Response 2

We are very grateful for this remark and fully agreed with your recommendation. In the revised manuscript, we carefully removed metaphorical or overly figurative formulations from Section 1 and replaced them with more precise and academically neutral language. In particular, the expression you highlighted was removed and the corresponding passage was reformulated in a more rigorous mechanistic style. More broadly, Section 1 was linguistically refined to ensure that the framing remains scientifically formal, conceptually clear, and fully aligned with the tone expected in an academic review article.

Comment 3

“It is recommended to shorten the descriptive part of section 1 somewhat, making the content of this section more concentrated and informative.”

Response 3

Thank you for this highly useful suggestion. We carefully revised Section 1 with the explicit goal of reducing descriptive excess and increasing conceptual density. Repetitive explanatory passages were removed or condensed, and the section was restructured so that each paragraph more directly advances a central mechanistic idea. The revised version is therefore shorter, more focused, and more informative, while preserving the key scientific framework necessary for the sections that follow.

Comment 4

“The authors illustrate section 2 well with a diagram. However, in the text of the review, they mention that ‘High-resolution optical and electron microscopy have shown a high density of KCC2 distribution in nanometer domains’ pp. 210–211. It would be very clear if the authors illustrated these data in the form of micrographs, with reference to the original sources.”

Response 4

We appreciate this thoughtful suggestion. We agree that direct visualization of these observations through representative micrographs could further strengthen the presentation. At the present stage, however, we chose not to add reproduced micrographs in order to avoid potential copyright and figure-permission complications and to maintain a coherent visual style across the review. Instead, we revised the corresponding text to make the evidence more explicit and clearly anchored to the original cited sources. We hope this approach preserves clarity while maintaining the structural consistency of the manuscript. We are nevertheless very grateful for this suggestion, which we found highly relevant.

Comment 5

“In section 2.1, the authors provide many descriptive characteristics. This section should be made more schematic and shortened.”

Response 5

Thank you very much. We fully agreed with this assessment and substantially revised Section 2.1 accordingly. The section was shortened, reorganized, and rewritten in a more schematic and concept-driven form. Rather than presenting multiple descriptive details sequentially, the revised text now emphasizes the core phospho-regulatory logic of KCC2, the major signaling inputs, and the principal functional implications in a more concise and structured manner. We believe this significantly improves readability and allows the main idea of the section to emerge more clearly.

Comment 6

“Section 2.3 should also be shortened by removing the redundant description. In particular, the information on pages 290–299, as well as 299–306, can be significantly reduced if the authors highlight the main ideas in this section. Due to the abundance of details, the authors do not always manage to convey the main idea to the readers.”

Response 6

We are very thankful for this careful and constructive observation. In response, we substantially condensed Section 2.3 and removed redundant descriptive material from the passages you indicated. We restructured the section around the principal conceptual axes of KCC2 failure—microdomain vulnerability, metabolic limitation, membrane destabilization, and maladaptive feedback amplification—so that the underlying logic of hierarchical collapse becomes more immediately visible. Our aim was to preserve mechanistic depth while making the section more accessible and more centered on its main ideas.

Comment 7

“The authors’ conclusion to section 2.3, pp. 317–325, should be made clearer and more readable.”

Response 7

Thank you very much. We agreed that the original concluding paragraph of Section 2.3 was overly dense and could be expressed more clearly. We therefore rewrote the conclusion in a more concise and readable form, while preserving its integrative function.

Comment 8

“In section 3.1, the authors should shorten the description on pages 335–343 and 344–353. Authors should not go into too much detail, artificially increasing the volume of the article; on the contrary, they should strive to find generalizations and present the material more concisely and constructively.”

Response 8

In response, we thoroughly revised Section 3.1 to reduce unnecessary detail and increase the level of synthesis. We believe this significantly improves the balance between mechanistic depth and readability.

Comment 9

“In section 3.2, the authors should shorten the description on pages 409–418. This and other sections of the review (see above) should be critically reviewed by the authors for generalization and reduction of details unrelated to the main idea of the section.”

Response 9

Thank you very much for this important broader comment. We fully agreed and revised Section 3.2 accordingly, condensing the text and highlighting the main developmental principles governing NKCC1 expression and chloride polarity maturation. In addition, we applied this same editorial principle more broadly across the manuscript, critically reviewing several sections to reduce secondary detail and improve conceptual concentration. This global revision substantially improved the coherence and proportion of the review.

Comment 10

“The authors presented a large amount of material in Table 1. It is necessary to title this table more clearly and less verbosely.”

Response 10

We are grateful for this observation. In response, we revised both the title and the structure of Table 1. The title was shortened and clarified, and the table itself was substantially redesigned to make it more compact, more visually accessible, and less text-heavy. We condensed the wording within the cells, reduced descriptive phrasing, and reorganized the content so that the table now functions more as a conceptual synthesis than as a block of extended text. We believe this improved both readability and overall presentation.

Comment 11

“The authors should review the content of section 5 according to the above scheme (reduce redundant descriptions, make necessary improvements, and possibly provide specific examples).”

Response 11

We carefully revised Section 5 in accordance with the same principles applied to the preceding sections. Redundant descriptions were removed, the discussion was made more compact and conceptually focused, and the main microcircuit consequences of chloride dysregulation were expressed with greater clarity. We also incorporated more explicit illustrative examples—such as the axon initial segment, dendritic plateau regulation, oscillatory regimes, and attractor destabilization—to anchor the discussion without reintroducing unnecessary verbosity. We believe this revision has made Section 5 substantially clearer and more informative.

Comment 12

“Sections 6 and 7, which provide and discuss specific pathological conditions of humans with impaired chloride metabolism, as well as therapeutic frontiers through microdomain engineering, are very useful and informative. The authors should carefully review and edit these sections again, reducing unnecessary descriptive details.”

Response 12

We are grateful for your positive assessment of Sections 6 and 7 and for this constructive suggestion. In response, we carefully re-edited both sections to reduce unnecessary descriptive detail while preserving their scientific scope and translational relevance. The revised versions are now more compact, denser, and more conceptually integrated. We removed repetitive phrasing, strengthened transitions, and focused each subsection more clearly on its central mechanistic and clinical message. We hope that these changes have improved the balance between informativeness and readability.

Once again, we would like to thank you for your careful evaluation of our manuscript and for the generous and constructive spirit of your comments. Your suggestions substantially improved the clarity, organization, and academic quality of the review. We are truly grateful for the time and expertise you invested in helping us strengthen this work.

With profound appreciation!

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Overall, the authors have improved the article's content in accordance with the comments made. The work has been shortened and improved and can be recommended for publication.

Comments on the Quality of English Language

English needs editing

Author Response

Dear Esteemed Academic Reviewer,

We thank you for your careful reassessment of our manuscript and for your kind and encouraging feedback.

We are grateful for your recognition of the improvements made in response to your previous comments. Your insightful suggestions have played an essential role in enhancing the clarity, structure, and overall quality of the work. We truly value the time and expertise you have dedicated to this review process.

We are honored by your recommendation for publication and remain sincerely appreciative of your thoughtful guidance throughout this process.

With our highest regards and gratitude!