Reserpine Causes Neuroendocrine Toxicity, Inducing Impairments in Cognition via Disturbing Hypothalamic–Pituitary–Thyroid Axis in Zebrafish

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The work by Sun et al. describes specific toxological effects of reserpine, an older antihypertensive drug that has some low, but significant use.  The work reads well and is performed with clear design and thurough data analysis. Formatting issues are the greatest limitation of this manuscript. 

Specifics include:

Small font in figures, blurred figures make the statistical significance (e.g. *** vs ****) a bit challenging to visualize.

There is an issue with a word or figure in the PDF generated for reviewers: "Error! Reference source not found" (in bold) occurs throughout the manuscript were the word "Figure" should be. 17 instances are observed.  This may occur at the author or publisher software.

References #1 and #5 are the same reference, but with a different format from the citation software used.  Please perform a detailed proof check and read.

Author Response

Comments 1: Small font in figures, blurred figures make the statistical significance (e.g. *** vs ****) a bit challenging to visualize.

Response 1: Thank the reviewer for pointing this out. We have adjusted the size of the font and resolution of the figures in the revised manuscript.

Comments 2: There is an issue with a word or figure in the PDF generated for reviewers: "Error! Reference source not found" (in bold) occurs throughout the manuscript were the word "Figure" should be. 17 instances are observed. This may occur at the author or publisher software (lines 202-297 in red).

Response 2: We apologize for these mistakes and have corrected them (lines 203-297 in red).

Comments 3: References #1 and #5 are the same reference, but with a different format from the citation software used. Please perform a detailed proof check and read.

Response 3: We apologize for this miswriting and have revised it.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript demonstrated that reserpine induces cognitive impairment in Zebrafish by causing neuroendocrine toxicity through damaging the hypothalamic-pituitary-thyroid axis. The work was well prepared and presented. However, I would like to provide some comments to improve the quality of the manuscript.

Comment 1: Distance travel data showed that 2 mg/L and 4 mg/L of reserpine exhibited reduced travel distance in both general and light and dark challenge distances (Fig 4. B and C). Could the author explain the reasons behind these reduced travel distances?

Comment 2: Gap expression generally increases in response to toxicity or inflammation. However, in the current study, reserpine dose-dependently decreased the gap expression. The author should include an explanation in the discussion.

Comment 3: From my understanding, reserpine started toxicity from 2 mg/L, but a significant c-fos expression was observed with only 16 mg/L. Can the author discuss this result?

 

Comments on the Quality of English Language

Nevertheless, the paper shows significant deficits in terms of grammar, leading to incorrect or unclear scientific descriptions in the paper. Examples: page 1, “As the results, the dose-dependent toxicity of reserpine on the neuroendocrine system (NES) had been revealed”

Author Response

Comments and Suggestions for Authors

Comments 1: Distance travel data showed that 2 mg/L and 4 mg/L of reserpine exhibited reduced travel distance in both general and light and dark challenge distances (Fig 4. B and C). Could the author explain the reasons behind these reduced travel distances?

Response 1: We thank the reviewer for this comment. This phenomenon may be due to the self-protective mechanisms underlying neurotoxic effects triggered by varying concentrations of reserpine [1]. When zebrafish treated with 2 mg/L and 4 mg/L of reserpine, a significant reduction in travel distance due to nervous system damage. As the reserpine concentration increased to 8 mg/L and 16 mg/L, cellular protective mechanisms might be activated, resulting in partial recovery of the nervous system. Despite there is the recovery, the decrease in travel distance of zebrafish remained statistically significant compared with the control (***p < 0.001) (lines 347-353 in red).

Comments 2: Gap expression generally increases in response to toxicity or inflammation. However, in the current study, reserpine dose-dependently decreased the gap expression. The author should include an explanation in the discussion.

Response 2: We thank the reviewer for this comment. Abnormal expression of gap43 is associated with various nervous system diseases. When nerves are damaged, the expression of this gene is upregulated to defend this injury. On the other hand, during development, insufficient expression of gap43 can impair synapse formation, leading to abnormal neural development. Previous studies have shown that olaquindox induces neurodevelopmental toxicity in zebrafish through the downregulation of gap43 expression. This is consistent with the results of our experiment (lines 373-379 in red).

Comments 3: From my understanding, reserpine started toxicity from 2 mg/L, but a significant c-fos expression was observed with only 16 mg/L. Can the author discuss this result?

Response 3: Thank the reviewer for this thoughtful comment. In our experimental investigation, a significant upregulation in expression levels was exclusively observed following exposure to elevated reserpine concentrations. This phenomenon may be attributed to that as an immediate-early gene, the expression of c-fos maybe slightly recovered after treatment with low concentrations of reserpine, while the highest of concentration 16 mg/L reserpine causes severe neural damage in zebrafish, leading to a significant increase in c-fos expression that cannot recover within a short period, demonstrating statistically significant differences (lines 387-394 in red).

Comments on the Quality of English Language

Comment: Nevertheless, the paper shows significant deficits in terms of grammar, leading to incorrect or unclear scientific descriptions in the paper. Examples: page 1, “As the results, the dose-dependent toxicity of reserpine on the neuroendocrine system (NES) had been revealed”.

Response: We apologize for the issues with grammar and unclear descriptions and greatly appreciate the reviewer's valuable comments. We have revised the whole manuscript (lines 18-19, 24-25, 53, 85-87, 142, 251-253, 362-364 and 442-443 in red).

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

 The manuscript “Reserpine Causes Neuroendocrine Toxicity, Inducing 2 Impairments in Cognition via Disturbing 3 Hypothalamic-Pituitary-Thyroid Axis in Zebrafish” by Sun et al. substantially improved after revision. The authors addressed all my concerns, so I recommend accepting the current version of the manuscript.