Review of Sethi et al

In the current study, Shivani S et al. explored the neural activation pattern of selected central nervous system (CNS) regions in the type 2 diabetes mellitus (DM) animal model. Zucker diabetic fatty (ZDF) rats were used as an animal model, and CNS regions that were known to affect the sympathetic nervous system directly were selected for analysis. Authors rationalized that identifying neural activation patterns in the central sympathoregulatory regions is necessary to understand the type 2 DM associated cardiac autonomic neuropathy. Authors used ΔFosB protein expression as a proxy for neuronal activation. In addition to the ΔFosB staining, authors did immunostaining for tyrosine hydroxylase and several neuropeptides. They showed evidence that there is an increase in the number of ΔFosB immunoreactive cells in the central sympathoregulatory regions of ZDF rats, and their phenotype is identified at some level.
The experimental design used in the current study might have enough potential to resolve the questions raised in the introduction. However, authors need to describe and clarify the methods used in the present study to support their findings and help other labs replicate them. These revisions are suggested as major and minor comments below.

Major comments

1. Authors should consider changing the article's title- from "sympathoregulatory brain regions and spinal cord" to "sympathoregulatory regions of the brain and spinal cord".
2. The conclusion statement in the abstract (lines 44-46) might be an over-interpretation of the presented data. Authors should consider changing it.
3. Does the manuscript need background information about proopiomelanocortin (POMC) cells in the introduction section?
4. According to lines 194-195, the authors did perfusion fixation on colchicine-treated rats. I am curious to know why the authors didn't conduct perfusion fixation on other rats (methods section 2.2.1 brains and spinal cord).
5. Did the authors use any rat brain atlas? If yes, please describe it in the methods.
6. What is the reason behind selecting three brain or spinal cord sections to analyze each area? Were the tissue sections sampled randomly for each area, or did any systemic sampling be used?
7. A table displaying section 3.1 numbers and statistics will be helpful for the reader.
8. Since the brain or spinal cord sections were not stained for Nissl, a description of how authors identified or defined each analyzed area on the tissue section is necessary. For example, the rectangular box identifying intermediolateral column (IML) in Figure 1B is more ventral to the spinal cord section, and it might not be IML.
9. In addition to the comment 8 description, authors should circumscribe the analyzed area on the images in Figures 1-7. This identification will help if other labs want to replicate the cell counts.
10. Authors suggested that increased activation of catecholaminergic neurons in the Rostral ventrolateral medulla can influence parasympathetic nerve activity (lines 300-303). Can authors elaborate on this interpretation?
11. Authors observed a decrease in the number of oxytocin immunoreactive cells in the paraventricular nucleus of the hypothalamus of ZDF rats (Figure 4C). Can authors discuss this result?
12. There are several mistakes in references; some of the errors were mentioned here.
13. References 8,15,28,40, and 48. Please change the journal name to "Journal of Comparative Neurology."
14. Reference 12. Please change the journal name to "Annals of Thoracic Cardiovascular Surgery."
15. Reference 19 and 35. Please change the journal name to "Proceedings of the National Academy of Sciences."
16. Reference 21. Please change the journal name to "Comprehensive Physiology."
17. I think reference 45 might be a book chapter. If yes, please format the reference.
    Authors should recheck all the references and correct them appropriately.
18. It is recommended that authors consult with antibodyregistry.com to include the appropriate RRID #s for each of the antibodies they used.

Minor comments

1. Authors might consider changing the running title.
2. Since the staining was done with an anti-TH antibody, authors should consider saying "Catecholaminergic-" instead of "noradrenergic-" (line 84).
3. It will be helpful for the novice readers if the authors can describe the dissection and sectioning of the spinal cord in methods section 2.2.1 or a reference might help.
4. If possible, can authors provide better images for Figure 1?
5. Please change mmol.L to mmol/L. If “mmol.L” is an accepted abbreviation, then ignore my comment.
6. If possible, authors should consider reporting the numbers and percentages in the results section as mean ± 95% confidence interval instead of mean ± standard error.
7. Scale bars in the figures can be made thinner so that they are less distracting