Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
In this manuscript, Chen et al. investigate the role of the membrane estrogen receptor GPR30 in spinal mechanisms of neuropathic pain. Using a wide variety of techniques, they first provide convincing evidence that GPR30 expression is restricted to neurons within the spinal cord, and that GPR30 neurons are well-positioned to receive descending input from the primary sensory cortex (S1). In addition, the authors put their findings in the context of the previous knowledge in the field, presenting evidence demonstrating that GRP30 is expressed in the majority of CCK-expressing spinal neurons. Overall, this manuscript furthers our understanding of neural circuity that underlies neuropathic pain and will be of broad interest to neuroscientists, especially those interested in somatosensation. Nevertheless, the manuscript would be strengthened by additional analyses and clarification of data that is currently presented.
Strengths:
The authors present convincing evidence for the expression of GPR30 in the spinal cord that is specific to spinal neurons. Similarly, complementary approaches including pharmacological inhibition and knockdown of GPR30 are used to demonstrate the role of the receptor in driving nerve injury-induced pain in rodent models.
Weaknesses:
Although steps were taken to put their data into the broader context of what is already known about the spinal circuitry of pain, more considerations and analyses would help the authors better achieve their goal. For instance, to determine whether GPR30 is expressed in excitatory or inhibitory neurons, more selective markers for these subtypes should be used over CamK2. Moreover, quantitative analysis of the extent of overlap between GPR30+ and CCK+ spinal neurons is needed to understand the potential heterogeneity of the GPR30 spinal neuron population, and to interpret experiments characterizing descending SI inputs onto GPR30 and CCK spinal neurons. Filling these gaps in knowledge would make their findings more solid.
Thank you very much for your constructive feedback.
In response to your suggestion, we have used more specific markers to distinguish excitatory (VGLUT2) and inhibitory (VGAT) neurons via in situ hybridization. These analyses revealed that GPR30 is predominantly expressed in excitatory neurons of the superficial dorsal horn (SDH), as presented in the Results section (lines 117-120) and in Figure 2A-B.
Additionally, we performed a quantitative analysis to determine the extent of co-localization between GPR30+ and CCK+ neurons. The data were included in the Results (lines 131–132) and Figure 2G.
Reviewer #2 (Public review):
Using a variety of experimental manipulations, the authors show that the membrane estrogen receptor G protein-coupled estrogen receptor (GPER/GPR30) expressed in CCK+ excitatory spinal interneurons plays a major role in the pain symptoms observed in the chronic constriction injury (CCI) model of neuropathic pain. Intrathecal application of selective GPR30 agonist G-1 induced mechanical allodynia and thermal hyperalgesia in male and female mice. Downregulation of GPR30 in CCK+ interneurons prevented the development of mechanical and thermal hypersensitivity during CCI. They also show the up modulation of AMPA receptor expression by GPR30.
Generally, the conclusions are supported by the experimental results. I also would like to see significant improvements in the writing and the description of results.
Methodological details for some of the techniques are rather sparse. For example, when examining the co-localization of various markers, the authors do not indicate the number of animals/sections examined. Similarly, when examining the effect of shGper1, it is unclear how many cells/sections/animals were counted and analyzed.
In other sections, there is no description of the concentration of drugs used (for example, Figure 4H). In Figures 4C-E, there is no indication of the duration of the recordings, the ionic conditions, the effect of glutamate receptor blockers, etc
Some results appear anecdotal in the way they are described. For example, in Figure 5, it is unclear how many times this experiment was repeated.
We sincerely appreciate your valuable feedback and thoughtful recommendations.
To address your concerns regarding methodological transparency, we have added the following details to the revised manuscript:
The number of animals and sections analyzed in co-localization studies.
The number of cells/sections/animals used in each quantification following shGper1 treatment.
The concentrations of drugs administered (e.g., in Figure 4H).
Detailed recording conditions, including duration, ionic composition, and pharmacological conditions (Figures 4C-E).
In addition, we have thoroughly revised the writing throughout the manuscript to enhance clarity and precision in the description of our findings.
Reviewer #3 (Public review):
Summary:
The authors convincingly demonstrate that a population of CCK+ spinal neurons in the deep dorsal horn express the G protein-coupled estrogen receptor GPR30 to modulate pain sensitivity in the chronic constriction injury (CCI) model of neuropathic pain in mice. Using complementary pharmacological and genetic knockdown experiments they convincingly show that GPR30 inhibition or knockdown reverses mechanical, tactile, and thermal hypersensitivity, conditioned place aversion, and c-fos staining in the spinal dorsal horn after CCI. They propose that GPR30 mediates an increase in postsynaptic AMPA receptors after CCI using slice electrophysiology which may underlie the increased behavioral sensitivity. They then use anterograde tracing approaches to show that CCK and GPR30 positive neurons in the deep dorsal horn may receive direct connections from the primary somatosensory cortex. Chemogenetic activation of these dorsal horn neurons proposed to be connected to S1 increased nociceptive sensitivity in a GPR30-dependent manner. Overall, the data are very convincing and the experiments are well conducted and adequately controlled. However, the proposed model of descending corticospinal facilitation of nociceptive sensitivity through GPR30 in a population of CCK+ neurons in the dorsal horn is not fully supported.
Strengths:
The experiments are very well executed and adequately controlled throughout the manuscript. The data are nicely presented and supportive of a role for GPR30 signaling in the spinal dorsal horn influencing nociceptive sensitivity following CCI. The authors also did an excellent job of using complementary approaches to rigorously test their hypothesis.
Weaknesses:
The primary weakness in this manuscript involves overextending the interpretations of the data to propose a direct link between corticospinal projections signaling through GPR30 on this CCK+ population of spinal dorsal horn neurons. For example, even in the cropped images presented, GPR30 is present in many other CCK-negative neurons. Only about a quarter of the cells labeled by the anterograde viral tracing experiment from S1 are CCK+. Since no direct evidence is provided for S1 signaling through GPR30, this conclusion should be revised.
Thank you for your encouraging comments and critical insights.
We fully acknowledge the concern regarding the proposed direct involvement of corticospinal projections in modulating nociceptive behavior via GPR30 in CCK+ neurons. While our anterograde tracing experiments suggest anatomical overlap, we agree that definitive evidence of functional connectivity is lacking.
Accordingly, we have revised the Abstract, Discussion, and Graphical Abstract to present our findings more cautiously. We now describe our observations as indicating that S1 projections potentially interact with GPR30+ spinal neurons, rather than asserting a definitive functional link.
To support this revised interpretation, we performed additional quantitative analyses examining the co-localization among S1 projections, CCK+, and GPR30+ neurons. Furthermore, we clarified that the chemogenetic activation studies targeted a mixed neuronal population and did not exclusively manipulate CCK+ neurons.
These changes aim to better align our conclusions with the presented data and provide a more nuanced framework for future investigations.
Reviewer #1 (Recommendations for the authors):
Major corrections
(1) Figure 2: The authors conclude that GPR30 is mainly expressed in excitatory spinal neurons because they are labeled by a virus with a Camk2 promoter. While there is evidence that Camk2 is specific to excitatory neurons in the brain, based on RNAseq datasets (e.g. Linnarsson Lab, http://mousebrain.org/adolescent/genesearch.html ) this is less clear cut within the spinal cord. A more direct way to assess the relative expression of GPR30 in excitatory versus inhibitory neurons would be to perform immunohistochemistry or FISH with GPR30/Vglut2/Vgat.
Alternatively, if this observation is not crucial for the overall arch of the story, I recommend the authors eliminate these data, as they do not support the idea that GPR30 is mainly in excitatory neurons.
We thank the reviewer for highlighting this important limitation. To strengthen our conclusion regarding the neuronal identity of GPR30-expressing cells, we performed fluorescent in situ hybridization (FISH) using vGluT2 (marker for excitatory neurons) and VGAT (marker for inhibitory neurons). The results confirmed that GPR30 is predominantly expressed in vGluT2-positive excitatory neurons within the spinal cord. These new data are presented in the revised manuscript (lines 117-120) and shown in Figure 2A-B.
(2) (2a) Figure 2: The authors also report that GPR30 is expressed in most CCK+ spinal neurons. A more rigorous way to present the data would be to perform quantification and report the % of CCK neurons that are GPR30.
(2b) More importantly, it is unclear what % of GPR30 neurons are CCK+. These types of quantifications would provide useful insights into the heterogeneity of CCK and GPR30 neuron populations, and help align findings of experiments using the behavioral pharmacology using GRP antagonists to the knockdown of Gper1 in CCK spinal neurons - for instance, does a population of GRP30+/CCK- neurons exist? If so, it would be worth discussing what role (if any) that population might play in nerve injury-induced mechanical allodynia.
Understanding the breakdown of GPR30 populations becomes even more relevant when the authors characterize which cell types are targeted by descending projections from S1. It is clear that the vast majority of CCK+ neurons that receive descending input from S1 neurons are GPR30+, but there are many other GPR30+ neurons that do not receive input from SI neurons presented in 5M. Is this simply because only a small fraction of CCK+/GPR30+ neurons are targeted by descending S1 projections, or could they represent a distinct population of GPR30 neurons?
(2a) We appreciate the suggestion. Quantification showed that approximately 90% of CCK⁺ neurons express GPR30, and about 50% of GPR30⁺ neurons co-express CCK. These data are now provided in the revised Results (lines 131-132) and in Figure 2F-G.
(2b) Indeed, our data reveal that a substantial portion of GPR30⁺ neurons do not co-express CCK. While this study focuses on GPR30 function in CCK⁺ neurons, we recognize the potential relevance of GPR30⁺/CCK⁻ populations. We have addressed this point in the Discussion (lines 303-306):
“However, it should be noted that half of GPR30⁺ neurons are not co-localized with CCK⁺ neurons, and further studies are needed to explore the function of these GPR30⁺/CCK⁻ neurons in neuropathic pain.”
Regarding descending input, our data in Figure 5 show that S1 projections selectively innervate a subset (~30%) of CCK⁺ neurons, most of which co-express GPR30. This suggests that S1-targeted CCK⁺/GPR30⁺ neurons may represent a functionally distinct population. We have added clarification to the revised manuscript, while acknowledging that further studies are needed to elucidate the roles of non-targeted GPR30⁺ neurons.
(3) Throughout the manuscript both male and female mice were used in experiments. Rather than referring to male and female mice as different genders, it would be more appropriate to describe them as different sexes.
As suggested, we have replaced all instances of “gender” with “sex” throughout the revised manuscript.
(4) Figure 5: To increase the ease of interpreting the figure, in panels 5J and 5N, it would be helpful to indicate directly on the figure panel which another marker was assessed in double-labeling analyses.
We have revised Figures 5J and 5N to include clear labels identifying the markers used in double-labeling analyses, to improve interpretability.
Minor corrections:
(1) Line 36, I believe the authors mean to say "GPER/GPR30 in spinal neurons", rather than just "spinal".
Corrected as suggested. The sentence now reads (line 34):
“Here we showed that the membrane estrogen receptor G-protein coupled estrogen receptor (GPER/GPR30) in spinal neurons was significantly upregulated in chronic constriction injury (CCI) mice…”
(2) There are minor grammatical errors throughout the manuscript that interfere with comprehension. Proofreading/editing of the English language use may be beneficial.
We have thoroughly revised the manuscript for clarity and corrected grammatical and syntactic errors to improve readability.
(3) Line 169-170, reads "Known that EPSCs are mediated by glutamatergic receptors like AMPA receptors and several studies have been reported the relationship between GPR30 and AMPA receptor25,29". Rewriting the sentence such that it better describes what the known relationship is between GPR30 and AMPA would be helpful in setting up the rationale of the experiment in Figure 4.
We have rewritten this section to better clarify the rationale behind the electrophysiological experiments (lines 161-164):
“Given that EPSCs are primarily mediated through glutamatergic receptors such as AMPA receptors, and emerging evidence suggesting that GPR30 enhances excitatory transmission by promoting clustering of glutamatergic receptor subunits, we examined whether GPR30 modulates EPSCs via AMPA receptor-dependent mechanisms.”
(4) Line 198-199 "Then we explored the possible connections among GPR30, S1-SDH projections and CCK+ neuron." In the context of spinal circuitry, "connections" may raise the expectation that synaptic connectivity will be evaluated. What I think best describes what the authors investigated in Figure 5 is the "relationship" between GPR30, S1-SDH projections, and CCK+ neurons.
We have revised the sentence accordingly (lines 184-186):
“Building on previous findings suggesting a functional interaction between S1-SDH projections and spinal CCK⁺ neurons, our current study aimed to further elucidate the structural relationship among GPR30, S1-SDH projections, and CCK⁺ neurons.”
(5) Figure 5: To increase the ease of interpreting the figure, in panels 5J and FN, it would be helpful to indicate directly on the figure panel which other marker was assessed in double-labeling analyses.
We have added direct labels to figure panels to clarify double-labeled analyses in the revised Figure 5J and 5N.
Reviewer #2 (Recommendations for the authors):
(1) Can the authors provide more detail about the distribution of CCK+ cells in the spinal cord and, in particular, the localization of double-stained (CCK/cfos) neurons?
We thank the reviewer for this suggestion. To better characterize the distribution of CCK⁺ neurons within the spinal dorsal horn (SDH), we performed immunostaining in CCK-tdTomato mice using lamina-specific markers: CGRP (lamina I), IB4 (lamina II), and NF200 (lamina III–V). Our results demonstrate that CCK⁺ neurons are primarily localized in the deeper laminae of the SDH. These findings are now described in the revised Results (lines 126–129) and shown in Figure 2E.
In addition, we conducted c-Fos immunostaining in CCK-Ai14 mice and found increased activation of CCK⁺ neurons following CCI. This supports the involvement of CCK⁺ neurons in neuropathic pain. These data are included in the Results (lines 129–131) and Supplementary Figure S4.
(2) Figure 2A. There is no formal quantification of the percentage of TdTomato+ neurons that are also CCK+. The description of these results is insufficient.
We appreciate this point and have revised the description of Figure 2A accordingly. To strengthen our analysis, we conducted additional FISH experiments with vGluT2 and VGAT probes. Quantification revealed that GPR30 is predominantly expressed in excitatory neurons (approximately 60%). These data are shown in the revised Results (lines 117-119) and Figures 2A-B and S3. This supports our conclusion that GPR30 is largely localized to excitatory spinal interneurons.
(3) Figure 4H. What is the evidence that these are AMPA-mediated currents? This is not explained in the text.
Thank you for raising this point. We now provide detailed experimental procedures to clarify that the recorded EPSCs are AMPA receptor–mediated. Specifically, spinal slices from CCK-Cre mice were used, and excitatory postsynaptic currents were recorded in the presence of APV (100 μM, NMDA receptor blocker), bicuculline (20 μM, GABA_A receptor blocker), and strychnine (0.5 μM, glycine receptor blocker), ensuring that the observed currents were AMPA-dependent. These methodological details are now clearly described in the revised Results (lines 165–173) and supported by prior literature (Zhang et al., J Biol Chem 2012; Hughes et al., J Neurosci 2010).
(1) Yan Zhang, Xiao Xiao, Xiao-Meng Zhang, Zhi-Qi Zhao, Yu-Qiu Zhang (2012). Estrogen facilitates spinal cord synaptic transmission via membrane-bound estrogen receptors: implications for pain hypersensitivity. J Biol Chem. Sep 28;287(40):33268-81.
(2) Ethan G Hughes, Xiaoyu Peng, Amy J Gleichman, Meizan Lai, Lei Zhou, Ryan Tsou, Thomas D Parsons, David R Lynch, Josep Dalmau, Rita J Balice-Gordon (2010). Cellular and synaptic mechanisms of anti-NMDA receptor encephalitis. J Neurosci. 2010 Apr 28;30(17):5866-75.
(4) What is the signaling mechanism leading to a larger amplitude of currents after G-1 infusion?
We thank the reviewer for this important question. G-1 is a selective agonist for GPR30. Based on previous studies by Luo et al. (2016), we speculate that activation of GPR30 may increase the clustering of glutamatergic receptor subunits at postsynaptic sites, thereby enhancing AMPA receptor-mediated currents. While our current study did not directly address the intracellular signaling cascade, we have incorporated this mechanistic speculation in the Discussion.
Jie Luo, X.H., Yali Li, Yang Li, Xueqin Xu, Yan Gao, Ruoshi Shi, Wanjun Yao, Juying Liu, Changbin Ke (2016). GPR30 disrupts the balance of GABAergic and glutamatergic transmission in the spinal cord driving to the development of bone cancer pain. Oncotarget 7, 73462-73472. 10.18632/oncotarget.11867.
(5) Figure 4I. Please include error bars.
We have revised Figure 4I to include error bars, as requested.
(6) Line 198. What is the evidence that AAV2/1 EF1α FLP is an antegrade trans monosynaptic marker?
We thank you for this request. AAV2/1 has been widely used for anterograde monosynaptic tracing based on its properties (Wang et al., Nat Neurosci 2024; Wu et al., Neurosci Bull 2021): (1) it infects neurons at the injection site and undergoes active anterograde transport; (2) newly assembled viral particles are released at synapses and infect postsynaptic partners; (3) in the absence of helper viruses, the spread halts at the first synapse, ensuring monosynaptic restriction. We have elaborated on this in the revised manuscript (line 198), citing Wang et al. (Nat Neurosci 2024) and Wu et al. (Neurosci Bull 2021).
(1) Hao Wang, Qin Wang, Liuzhe Cui, Xiaoyang Feng, Ping Dong, Liheng Tan, Lin Lin, Hong Lian, Shuxia Cao, Huiqian Huang, Peng Cao, Xiao-Ming Li (2024). A molecularly defined amygdalaindependent tetra-synaptic forebrain-tohindbrain pathway for odor-driven innate fear and anxiety. Nat Neurosci. 2024 Mar;27(3):514-526.
(2) Zi-Han Wu, Han-Yu Shao, Yuan-Yuan Fu, Xiao-Bo Wu, De-Li Cao, Sheng-Xiang Yan, Wei-Lin Sha, Yong-Jing Gao, Zhi-Jun Zhang (2021). Descending Modulation of Spinal Itch Transmission by Primary Somatosensory Cortex. Neurosci Bull. 2021 Sep;37(9):1345-1350.
(7) Figure 5G. I do not understand the logic of this experiment. A Cre AAV is injected in the S1 cortex. Why should this lead to the expression of tdTomato on a downstream (postsynaptic?) neuron? The authors should quote the literature that supports this anterograde transsynaptic transport.
We appreciate this question. As described in previous studies (e.g., Wu et al., Neurosci Bull 2021), AAV2/1-Cre injected into the S1 cortex leads to Cre expression in projection targets due to transsynaptic anterograde transport. Subsequent injection of a Cre-dependent AAV (AAV2/9-DIO-mCherry) into the spinal cord enables specific labeling of postsynaptic neurons that receive input from S1. We have clarified this mechanism in line 206 and provided the appropriate citation.
Zi-Han Wu, Han-Yu Shao, Yuan-Yuan Fu, Xiao-Bo Wu, De-Li Cao, Sheng-Xiang Yan, Wei-Lin Sha, Yong-Jing Gao, Zhi-Jun Zhang (2021). Descending Modulation of Spinal Itch Transmission by Primary Somatosensory Cortex. Neurosci Bull. 2021 Sep;37(9):1345-1350.
(8) The same question arises when interpreting the results obtained in Figure 6.
We thank the reviewer for the question, and we have addressed it in point (7).
(9) Line 257. How do the authors envision that estrogen would change its modulation of GPR30 under basal and neuropathic conditions? Is there any evidence for this speculation?
We thank the reviewer for raising this thoughtful question. In the current study, we focused on pharmacologically manipulating GPR30 activity via its selective agonist and antagonist. We did not directly investigate how endogenous estrogen regulates GPR30 under physiological and neuropathic states. We have recognized this limitation and highlighted the need for future research to investigate this regulatory mechanism.
(10-20) In my opinion, the entire manuscript needs a careful revision of the English language. While one can follow the text, it contains numerous grammatical and syntactic errors that make the reading far from enjoyable. I am highlighting just a few of the many errors.
We appreciate the reviewer’s honest assessment. The manuscript has undergone thorough language editing by a native English speaker to correct grammatical errors, improve clarity, and enhance overall readability. We also restructured several sections, particularly the Discussion, to improve logical flow.
(21) The discussion of results is a bit disorganized, with disconnected sentences and statements, and somewhat repetitive. For example, lines 303 to 306 lack adequate flow. It is also quite long and includes general statements that add little to the discussion of the new findings (lines 326-333).
We agree and have revised the Discussion extensively. Disconnected or repetitive sentences (e.g., lines 303-306, 326-333) have been removed or rewritten. For instance, we added a new transitional paragraph (lines 307-311) to improve flow:
“Abnormal activation of neurons in the SDH is a key contributor to hyperalgesia, and enhanced excitatory synaptic transmission is a major mechanism driving increased neuronal excitability. Therefore, we evaluated excitatory postsynaptic currents (EPSCs) and observed increased amplitudes in CCK⁺ neurons following CCI, suggesting elevated excitability in these neurons.”
We also removed redundant generalizations to maintain a focused discussion of our novel findings.
Reviewer #3 (Recommendations for the authors):
(1) What is the distribution of GPR30 throughout the spinal cord and DRG? The authors demonstrate that this can overlap with a CCK+ population, but there are many GPR30+ and CCK negative neurons, even in the cropped images presented. It would be helpful to quantify the colocalization with CCK.
We thank the reviewer for this important point. As shown in the revised manuscript, GPR30 is expressed in both the spinal cord and dorsal root ganglia (DRG). However, our updated data (Figure 1B) demonstrate that Gper1 mRNA levels in the DRG are not significantly altered after CCI, suggesting a limited involvement of DRG GPR30 in neuropathic pain. These results are described in the revised Results (line 94).
Regarding spinal co-expression, we performed a detailed quantification. Approximately 90% of CCK⁺ neurons express GPR30, while about 50% of GPR30⁺ neurons are CCK⁺. These co-localization results are now included in the revised Results and presented in Figure 2G.
(2) It is clear that CCI and GPR30 influence excitatory synaptic transmission in CCK+ neurons. However, these experiments do not fully support the authors' claims of a postsynaptic upregulation of AMPARs. Comparing amplitudes and frequencies of spontaneous EPSCs cannot necessarily distinguish a pre- vs postsynaptic change since some of these EPSCs can arise from spontaneous action potential firing. I suggest revising this conclusion.
We appreciate these insightful comments. We fully agree that our data from spontaneous EPSC recordings (sEPSCs) in CCK⁺ neurons are not sufficient to distinguish between pre- and postsynaptic mechanisms, as sEPSCs may include spontaneous presynaptic activity. Therefore, we have revised the text throughout the manuscript to avoid overstating conclusions related to postsynaptic AMPA receptor upregulation.
(3) What is the rationale for the evoked EPSC experiments from electrical stimulation in "the deep laminae of SDH?" I do not think that this experiment can rule out a presynaptic contribution of GPR30 to the evoked responses, particularly if these are Gs-coupled at presynaptic terminals. Paired-pulse stimulations could help answer this question, otherwise, alternative interpretations, also related to the point above, should be provided.
We thank the reviewer for this thoughtful critique. Indeed, electrical stimulation of the deep SDH laminae does not exclude presynaptic involvement, especially considering that GPR30 is a G protein–coupled receptor (GPCR) and could act presynaptically. We agree that paired-pulse ratio (PPR) analysis would be more informative in distinguishing pre- from postsynaptic effects, but this was not performed due to technical limitations in our current experimental setup.
Accordingly, we have revised our interpretations in both the Results and Discussion to acknowledge that our data do not rule out presynaptic contributions. We now state that GPR30 activation enhances EPSCs in CCK⁺ neurons, while further studies are needed to dissect the precise site of action.
(4) I appreciate the challenging nature of the trans-synaptic viral labeling approaches, but the chemogenetic and Gper knockdown experiments do not selectively target this CCK+ population of deep dorsal horn neurons. The data are clear that each of these components (descending corticospinal projections, CCK neurons, and GPR30) can modulate nociceptive hypersensitivity, but I do not agree with the overall conclusion that each of are directly linked as the authors propose. I recommend revising the overall conclusion and title to reflect the convincing data presented.
We thank the reviewer for this critical observation. We agree that while our data show functional roles for descending cortical input, CCK⁺ neurons, and GPR30 in modulating pain hypersensitivity, the evidence does not establish a definitive direct circuit integrating all three components.
In response, we have revised our conclusions to reflect this limitation. Specifically, we avoided claiming a direct functional link among S1 projections, CCK⁺ neurons, and GPR30. Instead, we now propose that GPR30 modulates neuropathic pain primarily through its action in CCK⁺ spinal neurons, with potential involvement of descending facilitation from the somatosensory cortex.
Additionally, we have revised the manuscript title to better reflect our mechanistic focus:
“GPR30 in spinal CCK-positive neurons modulates neuropathic pain.”
Minor Corrections
(1) The authors should refer to mice by sex, not gender.
Corrected throughout the manuscript.
(2) Page 9, line 195: "significantly" is used to refer to co-localization of 28.1%. What is this significant to?
We have revised the sentence to accurately describe the observed percentage, without implying statistical significance:
“Our co-staining results revealed that a high proportion of CCK⁺ S1-SDH postsynaptic neurons expressed GPR30” (line 198-199).
(3) I recommend modifying some of the transition phrases like "by the way," "what's more," and "besides".
All informal expressions have been replaced with academic alternatives including “Furthermore,” “Additionally,” and “Moreover.”
(4) Additional guides to mark specific laminae in the dorsal horn would be useful.
We added immunostaining with laminar markers (CGRP for lamina I and NF200 for lamina III–V), and these data are now shown in Figure 2E and described in the Results (lines 126-129).
(5) Page 5, line 115: immunochemistry should be immunohistochemistry.
Corrected as suggested.
(6) Page 6, line 136: "Confirming the structural connnections" was not demonstrated here. Perhaps co-localization between GPR30 and CCK+.
The text was revised to “To functionally interrogate GPR30 and CCK⁺ neurons in neuropathic pain...” (line 133).
(7) Page 8, line 166: unsure what "took and important role" means.
This phrasing was corrected for clarity and replaced with an accurate scientific description.
(8) Page 8, line 168: "IPSCs of spinal CCK+ neurons" implies that they are sending inhibitory inputs.
We revised the term to “EPSCs” to correctly reflect excitatory synaptic currents in CCK⁺ neurons.
(9) Page 8, line 169: "Known that EPSCs" is missing an introductory phrase.
The sentence was rewritten to include an appropriate introductory clause (lines 161–164):
“Given that EPSCs are primarily mediated through glutamatergic receptors such as AMPA receptors...”
(10) Page 10, line 227 and 228: "adequately" and "sufficiently" should be adequate and sufficient.
We corrected these terms to the proper adjective forms: “adequate” and “sufficient” (lines 224-225).
