Gustafson et al. further investigated the role of prolactin in the suppression of the hypothalamic-pituitary-adrenal (HPA) axis during lactation. They employed different methodologies in virgin and lactating mice, such as in situ hybridization (CRH), corticosterone and prolactin assays, immunohistochemistry (pSTAT5), and RNA scope (prolactin receptor), which confirm previous findings and expand the knowledge on the maternal adaptations of the stress responses. The subject is of interest and the authors have expertise in the field of research. Overall, The work is properly justified and the manuscript is well prepared. I have a few concerns and comments to be addressed by the authors.
1) Although I think the original findings bring new information to the field, this is not objectively and sufficiently stated in the manuscript. For example, the Title seems a bit too vague concerning the work’s findings, and the same happens with the conclusions provided in the Abstract and Discussion. Thus, the authors should clearly describe and emphasize the novelty of their results to the field of research.
2) Line 58. Please, check the citation of reference 4, which seems to be in mice and not rats.
3) Lines 63 and 363. Check citations of reference 13, which does not seem fully related to the text in these places.
4) Line 71. Reference 13 is a duplicate of reference 5.
5) Materials and methods/Animals. This is a confusing part of the manuscript because it is not sufficiently clear which animals were used in each analysis and what times were used for euthanasia or sample collection in each case. It is important to identify when the same group of animals was used in different analyses and when not. The authors should also clearly describe for each analysis the time points used for tissue collection regarding the time of the day, lactation (suckling), and restraint stress. For example, in line 118, According to the assumption from the description provided in this paragraph, trunk blood for corticosterone measurement would have been collected around 11:00 h, in contrast with the description that “…All animals were terminated between 1200 h and 1320 h”.
6) Corticosterone Elisa. There is little information about this assay in the manuscript and no reference to a previous use by the authors. It is not clear which sort of samples were used, dilution factor, sensitivity, variability, and if the assay effectiveness in mice was validated.
7) Figure 1. The boxes indicating the magnified areas and scale bars are missing in 1B. Examples of detected neurons positive to CRH might be indicated.
8) Figure 3. Unlike the other figures, prolactin levels were obtained as temporal paired data that would be better shown as a line graph. Columns might show integrated levels. Define PRL in y-axis.
9) Figure 4. Please check the unit of scale bars, is mm correct? Also, the N value described in the legend does not match the number of individual points in the columns.
10) It is intriguing that pSTAT5 immunoreactivity in the adrenal cortex did not differ between V, L, and L/S mice despite the marked differences in prolactin levels. In the Discussion, the authors argue that this can be due to a combination of lower receptor expression and modifications in the prolactin receptor-signaling pathway in the lactating adrenal. Although this is a possibility, can one be sure that the increased pSTAT5 in the adrenal of stressed virgin mice is an effect of prolactin and not growth hormone? Ideally, data showing the effects of prolactin and/or bromocriptine would be required. If that is not feasible, this caveat should be discussed in the manuscript.
11) Figure 5. The data on prolactin receptor expression in the adrenal cortex of virgin and lactating mice are really interesting. However, it is not clear how the labelings were quantified, was it the density of pixels or number of grains? This should be described in more detail. Moreover, I suggest including an image of the RNA scope negative control to make clear what is the absence of labeling. In this respect, the data of prolactin receptor mRNA levels in the medulla are shown (5C) but, at the same time, described as indistinguishable from the background in the text and Figure Legend. This should be revised for consistency, was it expressed or not in the medulla?
12) Line 405. The interpretation “The complete absence of pSTAT5 positive cell nuclei within the adrenal cortex in stressed lactating mice made in the current study…’’ is not supported by the counting of pSTAT5 positive cells shown for lactating and lactating/stress mice in figure 4G. This should be revised for consistency.

The manuscript was notably improved in this revised version. Although I recognize that the authors did well in answering the reviewers’ comments and improving the manuscript, I disagree with their response to one of my comments, and there appear to be a couple of new points to be addressed in the manuscript. Please find the specific comments below.
Authors’ answer to referee2:
7. In response to concerns raised by this referee the time window of blood collection for CORT determination has now been included in the above section as "between 1200h and 1230 h" for all animals.
The information "between 1200h and 1230 h" is not found in the revised manuscript.
8. ‘’…Briefly, duplicate samples from unstressed (10 μl) and stressed (5 μl) diestrus and lactating mice were prepared with a dissociation reagent and assay buffer to a final dilution of 1:100 and 1:50, respectively…".
It is not clear in the rewritten text which experimental condition (unstressed, stressed, diestrus, and lactating) is referred to by "respectively". Please, clarify.
11. While the referee is correct that Figure 3 contains temporal information regarding prolactin levels, we believe that the widely spaced time points of 7 and 30 min make it inappropriate to construct a "line graph" because we have no information of any fluctuations in prolactin levels between these two time points.
I do not agree with the authors’ response to this question. The "spaced time points" do not justify the use of a column graph for this data set. A line graph is the correct format. Moreover, the statistical analysis is not clearly described in this figure (3). Is it a two-way ANOVA with repeated measures? If so, the results of the text should be described in more detail, and the respective symbols of significance should be included in the figure.