Psychosocial Stress Induces Orofacial Mechanical Allodynia Due to the Enhancement of Transient Receptor Potential Ankyrin 1 Expression in Trigeminal Ganglion Neurons via the Increment of the Trace Amine-Associated Receptor 7f Expression

Round 1

Reviewer 1 Report

In this study, which is entitled “Psychosocial stress induces orofacial mechanical allodynia due to the enhancement of transient receptor potential ankyrin 1 expression in trigeminal ganglion neurons via the increment of the trace amine associated receptor 7f expression”, authors conducted chronic psychosocial stress experiments in mice induced the orofacial mechanical allodynia through enhancement of TRPA1 expression in trigeminal ganglion neurons with changes in the levels of trace amine-associated receptor 7f.

Study design has novelty and results are important in this field.

To strengthen this article more, here a reviewer suggests several points as below.

Authors compared CfH and SiH groups in animal study. These both words first appear in results (L74-75). Authors should show the spelled out in the first time. In the current version, spell out is seen in L162-163 in materials and methods. 

In Figure 3, data is striking. Authors compared the number of TRPA1-positive neurons between CfH group and SiH group. To confirm this data, at least authors need to show immunostaining pictures data of each group, which shows significant difference. And authors need to discuss why the number of “medium-sized” TRPA1-positive trigeminal ganglion neurons were significantly higher in the CfH group than in the SiH group.

In addition to TRPA1, other several types of TRP channels are likely to contribute to orofacial mechanical allodynia. Authors should discuss the possibility of other mechanosensitive ion channels and distinct different properties of TRPA1 in comparison to others in this study and orofacial mechanical allodynia field.

Author Response

We thank the Reviewer for their insightful and helpful comments. We have tried to address all issues raised by the reviewers. Also, we have thoroughly revised the manuscript and adding new figures. We highlighted the corrected or additional words/sentences with red font and the underline.

Reviewer #1: In this study, which is entitled “Psychosocial stress induces orofacial mechanical allodynia due to the enhancement of transient receptor potential ankyrin 1 expression in trigeminal ganglion neurons via the increment of the trace amine associated receptor 7f expression”, authors conducted chronic psychosocial stress experiments in mice induced the orofacial mechanical allodynia through enhancement of TRPA1 expression in trigeminal ganglion neurons with changes in the levels of trace amine-associated receptor 7f.

Study design has novelty and results are important in this field.

To strengthen this article more, here a reviewer suggests several points as below.

Authors compared CfH and SiH groups in animal study. These both words first appear in results (L74-75). Authors should show the spelled out in the first time. In the current version, spell out is seen in L162-163 in materials and methods.

Author’s Response: Thank you very much for the comments. We have collected these words (CfH and SiH) properly as follows. “The level of CYP11B1 in the confrontational housing (CfH) group was significantly lower than that in the single housing (SiH) group…”. (line 73-74)

In Figure 3, data is striking. Authors compared the number of TRPA1-positive neurons between CfH group and SiH group. To confirm this data, at least authors need to show immunostaining pictures data of each group, which shows significant difference. And authors need to discuss why the number of “medium-sized” TRPA1-positive trigeminal ganglion neurons were significantly higher in the CfH group than in the SiH group.

Author’s Response: Thank you very much for the important suggestions. We have added immunostaining pictures as Fig. 3B and the figure legend. We have also added the sentences as follows in Results. “We also showed the immunostaining of TRPA1-positive trigeminal ganglion neurons in both groups (Fig. 3B).” (line 95-96)

In addition to TRPA1, other several types of TRP channels are likely to contribute to orofacial mechanical allodynia. Authors should discuss the possibility of other mechanosensitive ion channels and distinct different properties of TRPA1 in comparison to others in this study and orofacial mechanical allodynia field.

Author’s Response: Thank you very much for the valuable comments. We have added the following sentences in Discussion. “Other types of TRP channels are associated with orofacial allodynia [22]. Transient receptor potential vanilloid 1 (TRPV1) depletion in genetic mice reveals remission of mechanical allodynia and the activation of TRPV1 induces mechanical allodynia [23]. Inhibition of transient receptor potential vanilloid 4 (TRPV4) attenuates mechanical allodynia in a rat model of intraoral wire-induced mucositis [24]. The inhibition of transient receptor potential melastatin 8 (TRPM8) with capsazepine significantly reduces cold pain, suggesting that TRPM8 may play a role in cold allodynia [25].” (line 129-135)

Reviewer 2 Report

Stresses 2108762

The study appears interesting, methodologically well structured and conducted. The methods used are adequate and the statistical analysis sufficient.

However, there are some problems and limitations, also recognized by the authors themselves, which make acceptance of the conclusions problematic.

The major criticisms are listed below

Line 148 and following: This sentence is crucial: the data collected seem to suggest this conclusion. On the other hand, the sentence should be integrated with a doubtful observation, and the note of line 148 (study limitations) should be added. It is necessary to unequivocally define that the upregulation of TAAR7f is linked to psychosocial stress and not to physical stress is fundamental and severely limits the interpretation of the data collected in this work.

Line 241: Conclusion session needs to be expanded and rewritten. It is necessary to report the obvious limitations of the research and the partiality of the data collected which cannot be generalized.

Minor concerns are:

Line 68: The Results section should be moved between the Material & Methods and Discussion sections

Figure 2: the asterisks of statistical significance are not shown in the graph of figure 2

Line 110 : The Discussion section should be moved after the Materials and Methods

Lines 139-141: This sentence is very interesting, but it is also mostly speculative. It would be necessary to broaden the discussion on this point which is crucial to give meaning to the research.

Line 170: This paragraph needs to be expanded to allow for a full assessment of the methodology employed

Author Response

We thank the Reviewer for their insightful and helpful comments. We have tried to address all issues raised by the reviewers. Also, we have thoroughly revised the manuscript and adding new figures. We highlighted the corrected or additional words/sentences with red font and the underline.

Review Comments to the Author

Reviewer #2:

The study appears interesting, methodologically well structured and conducted. The methods used are adequate and the statistical analysis sufficient.

However, there are some problems and limitations, also recognized by the authors themselves, which make acceptance of the conclusions problematic.

The major criticisms are listed below

Line 148 and following: This sentence is crucial: the data collected seem to suggest this conclusion. On the other hand, the sentence should be integrated with a doubtful observation, and the note of line 148 (study limitations) should be added. It is necessary to unequivocally define that the upregulation of TAAR7f is linked to psychosocial stress and not to physical stress is fundamental and severely limits the interpretation of the data collected in this work.

Author’s Response: Thank you very much for the valuable comments. We strongly agreed your opinion. We added the following sentences in Discussion. “Finally, the relationship between TAAR7f and psychosocial stress still remains unclear in this study.” (line 164-165)

Line 241: Conclusion session needs to be expanded and rewritten. It is necessary to report the obvious limitations of the research and the partiality of the data collected which cannot be generalized.

Author’s Response: Thank you very much for the comments. We have added the following sentences in Conclusion. “However, because of the many limitations of this study, it is difficult to conclude whether chronic psychosocial stress induces an increase in TAAR7f expression in the blood via TRPA1. For example, it is necessary to show that chronic psychosocial stress does not up-regulate blood TAAR7f expression in mice lacking TRPA1.” (line 272-275)

Minor concerns are:

Line 68: The Results section should be moved between the Material & Methods and Discussion sections

Author’s Response: Thank you very much for the comments. However, this journal demands the order of “Results”, “Discussion” and “Materials & Methods”.

Figure 2: the asterisks of statistical significance are not shown in the graph of figure 2

Author’s Response: Thank you very much for the comments. We have added the asterisks in Figure 2.

Line 110 : The Discussion section should be moved after the Materials and Methods

Author’s Response: Thank you very much for the comments. However, this journal demands the order of “Results”, “Discussion” and “Materials & Methods”.

Lines 139-141: This sentence is very interesting, but it is also mostly speculative. It would be necessary to broaden the discussion on this point which is crucial to give meaning to the research.

Author’s Response: Thank you very much for the comments. We have changed the sentences in Discussion as follows. “We propose the following hypothesis by which trace amine induces orofacial pain. Trace amines in blood induced by psychosocial stress bind to the TAAR7f expressed by trigeminal ganglion neurons, resulting in increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 since activation of TAARs is induced through ERK1/2 pathway [35]. Phosphorylation of ERK1/2 induces facial pain because it promotes the ex-pression of TRPA1 protein [36].” (line 150-155)

Line 170: This paragraph needs to be expanded to allow for a full assessment of the methodology employed

Author’s Response: Thank you very much for the comments. We have changed the sentences in Materials and Methods as follows. “Two mice were housed separately in the cage for 7 days with one mouse in each subunit. The mice could not look at or be in contact with each other as a result of the partition (Fig. 5A and 5B). And then the partition was removed to expose the mice to confrontational stress as shown in Fig. 5C.” (line 182-186). And we also added Figure 5 and the figure legend.

Round 2

Reviewer 1 Report

Well addressed

Reviewer 2 Report

The paper in the present version can be accepted