Modulation of dorsal premotor cortex differentially influences I‐wave excitability in primary motor cortex of young and older adults

Abstract Previous research using transcranial magnetic stimulation (TMS) has demonstrated weakened connectivity between dorsal premotor cortex (PMd) and motor cortex (M1) with age. While this alteration is probably mediated by changes in the communication between the two regions, the effect of age on the influence of PMd on specific indirect (I) wave circuits within M1 remains unclear. The present study therefore investigated the influence of PMd on early and late I‐wave excitability in M1 of young and older adults. Twenty‐two young (mean ± SD, 22.9 ± 2.9 years) and 20 older (66.6 ± 4.2 years) adults participated in two experimental sessions involving either intermittent theta burst stimulation (iTBS) or sham stimulation over PMd. Changes within M1 following the intervention were assessed with motor‐evoked potentials (MEPs) recorded from the right first dorsal interosseous muscle. We applied posterior–anterior (PA) and anterior–posterior (AP) current single‐pulse TMS to assess corticospinal excitability (PA 1mV ; AP 1mV ; PA 0.5mV , early; AP 0.5mV , late), and paired‐pulse TMS short intracortical facilitation for I‐wave excitability (PA SICF, early; AP SICF, late). Although PMd iTBS potentiated PA 1mV and AP 1mV MEPs in both age groups (both P < 0.05), the time course of this effect was delayed for AP 1mV in older adults (P = 0.001). Furthermore, while AP 0.5mV , PA SICF and AP SICF were potentiated in both groups (all P < 0.05), potentiation of PA 0.5mV was only apparent in young adults (P < 0.0001). While PMd influences early and late I‐wave excitability in young adults, direct PMd modulation of the early circuits is specifically reduced in older adults. Key points Interneuronal circuits responsible for late I‐waves within primary motor cortex (M1) mediate projections from dorsal premotor cortex (PMd), but this communication probably changes with advancing age. We investigated the effects of intermittent theta burst stimulation (iTBS) to PMd on transcranial magnetic stimulation (TMS) measures of M1 excitability in young and older adults. We found that PMd iTBS facilitated M1 excitability assessed with posterior–anterior (PA, early I‐waves) and anterior–posterior (AP, late I‐waves) current TMS in young adults, with a stronger effect for AP TMS. M1 excitability assessed with AP TMS also increased in older adults following PMd iTBS, but there was no facilitation for PA TMS responses. We conclude that changes in M1 excitability following PMd iTBS are specifically reduced for the early I‐waves in older adults, which could be a potential target for interventions that enhance cortical excitability in older adults.

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----------------EDITOR COMMENTS Reviewing Editor: Thank you for submitting your manuscript to the Journal of Physiology where it has been carefully reviewed by 2 expert reviewers and by the editorial team.As you can see, while all appreciate the question you ask and are broadly positive about the paper and its potential impact on the field, both reviewers have a number of comments that will need to be addressed.
In addition, the editorial team make the following points: The key points and title are appropriate.The ToC category is correct.The reviewers raise a few points that would benefit from some clarification, but broadly the introduction and methods are clear and well written.The figures show individual data points and n is clear -thank you.The authors use a 95% Confidence Intervals rather than Standard Deviation throughout but this is clearly stated and appropriate.The figures and tables are clear.The manuscript is well written.

REFEREE COMMENTS
Referee #1: This is a very nice paper, nice work and clearly explained.I have a few queries for the authors: What, more specifically, does "low-level activation of the right FDI" refer to?Rejection of trials with baseline EMG activity above 25uV is mentioned later on, so was AMT recorded during contraction with maximum amplitudes of of 25uV?"Following AMT, the stimulus intensities producing a standard MEP amplitude approximating 1 mV (MEP1mV; PA1mV and AP1mV), in addition to an MEP amplitude approximating 0.5 mV (MEP0.5mV;PA0.5mV and AP0.5mV), when averaged over 20 trials, were identified" -What was the procedure for this?For example, was the stimulator intensity systematically varied after averaging 20 trails at each intensity?Was PEST used?"For all measures of SICF, individual MEP amplitudes produced by paired-pulse stimulation were expressed as a percentage of the mean MEP amplitude produced by single-pulse stimulation recorded at baseline (Cash et al., 2009;Liao et al., 2022), as Spearman's correlation analysis revealed that post-intervention increases in single-pulse test MEP amplitudes predicted increases in the paired-pulse MEP amplitudes (ρ = 0.8, P < 0.05)" -I'm confused by this approach.Say for example that iTBS increases single pulse MEP amplitude and increases paired-pulse MEP amplitude, should taking the conditioned vs unconditioned average MEP amplitude ratio not correct for this?By comparing pre-iTBS single pulse with post-iTBS paired pulse are you not then incorporating the effect of iTBS on single pulse MEPs into your SICF measure?"Correlations are presented as Spearman's ρ with significance set at P < 0.00167 following Bonferroni correction."-Why use Bonferroni?At a correction across 30 comparisons to achieve a corrected P threshold of 0.00167 you are likely overcorrecting.What about adaptive FDR?
The use of the term "low-intensity" is confusing/misleading, I presume this refers to the use of peri-threshold intensities, but it could be interpreted as sub-threshold or as low intensity absolute values (for example <30% MSO) What did participants do in the 1 hour intervals before and after iTBS and in the gap between 5 40 min post-iTBS measurements?
"We found that both PA SICF1.5 and AP SICF4.5 were potentiated (~20-40% increase) across both young and older adults.As the 1.5ms ISI assesses early I-waves and 4.5 ms ISI assesses late I-waves (Ziemann et al., 1998;Opie et al., 2018), one interpretation of these results could be that PA SICF1.5 activates PA early I-waves, whereas AP SICF4.5 activates AP late I-waves.This would be consistent with our single-pulse findings for PA0.5mV and AP0.5mV, and suggests that PMd iTBS may preferentially modulate both PA early and AP late I-waves."-Two points on this.First, I think the authors should remove the reference to "early PA" and late AP" throughout, as it implies that PA only activates early I waves, and AP only activates late I waves, in addition to suggesting that the late I waves activated by one orientation are different from he late I waves activated by another (ditto for early I waves).It would be better to simply refer to early or late I waves when naming the I waves.Second, it is later mentioned that "it is possible that the measure of SICF was complicated by the MEP1mV test stimulus, which likely resulted in mixed recruitment of I wave circuits (Opie et al., 2021)", which is a good point.Could the detection of an effect on SICF4.5ms using AP and on SICF1.5ms using PA reflect that these different orientations preferentially (but not solely) activate the I waves that are intended to be interrogated by these different ISIs, and therefore there is more to be facilitated/inhibited.For example, if AP orientation engages late I wave circuits moreso than AP orientation, then it seems logical that AP orientation would be a better orientation to use when attempting to facilitate the late I waves with SICF4.5ms, and would be more sensitive to effects of iTBS on said measure.
Overall I think there needs to be a bit of caution regarding intermixing reference to early vs late I waves and AP vs PA orientation, as at suprathreshold intensities such as those evoking 1mV MEPs, both I waves will be engaged by both orientations.More emphasis on "preferential" engagement is warranted.

Referee #2:
Comments on Liao et al.Liao et al. investigated the effects of PMd iTBS on MEP amplitudes and latencies with both PA and AP currents as well as 0.5mV and 1mV TS.The investigators also examined PA and AP effet for SICF at 1.5 and 4.5 ms ISIs.Overall, younger adults had higher MEP amplitudes, shorter MEP latencies and higher stimulation intensities for PA at 0.5 and 1mV.The main findings related to iTBS were significant increases iTBS compared to sham, with the only prominent group difference that older adults failed to demonstrate a significance increase compared to sham at 5 minutes post (but younger adults did).
Overall, the manuscript was very well written, easy to follow and examined an interesting research question that was supported by a hypothesis.The majority of my comments are relatively minor and aimed at improving the reader's understanding and readability of the manuscript.

Comments:
Although Figure 1 alludes to it, it is not clearly stated in text that the left PMd was always targeting.Please make this clear in the methods section for iTBS In the data analysis section you state "as Spearman's correlation analysis revealed that post-intervention increases in single-pulse test MEP amplitudes predicted increases in the paired-pulse MEP amplitudes (ρ = 0.8, P < 0.05)".Are these results from your work or the previous work?If your work, please include them in the results section.
A visual from BrainSight for the stimulation location of PMd for all participants on standard MNI-ICBM152 template as well as a table with MNI coordinates (this could be supplemental figures and table).I think this is particularly important since PMd location was not based off function or structure from individualized MRIs.
In the data analyses, you collapsed the MEP latency data across multiple stimulation intensities.Please provide data (even in supplemental data) that there were no differences in MEP latency across PA1mV, AP 1mVn PA0.5mV and AP0.5mV.
Why did the analyses investigating the changes in corticospinal excitability following the intervention using baselinenormalized values not look at the PA1mV, AP 1mVn PA0.5mV and AP0.5mV altogther?This is the statistical method you used for the SICF measures.
In the analyses, you stated you used Spearman's rank-order correlation, but then stated you regressed variables.Did you correlate two variables (which is what Spearman's rank-order correlation does) or regress?If you did a regression, please state proper analyses.
Due to the inability to record several of the measures, it would be important for the authors to include which participants (example participant code) and their demographic variables were excluded, and the remaining demographic variables for those left in the analyses (again, this is something that could be put in supplemental data).I believe it is interesting to the readers to understand what may have been different about these participants and why might that have contributed to not being able to record these measures.
I am curious why you also didn't include PA1mV over time in young vs. old within Figure 2? I know there was not statistically significant difference, but it provides a nice visual contrast to the AP1mV already included in Figure 2 (part C) Older adults failed to demonstrate they were different with iTBS compared to sham with PA0.5mV but did demonstrate for AP0.5mV.When taken into consideration with the older adults only demonstrating a significant increase in AP1mV 40 (vs.5) minutes post, what can that all mean?Are these changes correlated?
No differences between young vs. older adults for SICF.PA1.5mV increased at 40 minutes and compared to sham while AP4.5mV increased after iTBS compared to sham.APSICF1.5 did not change but there was no discussion of PA4.5mV results?Please include a statement regarding PA4.5mV SICF.
In regards to Figure 5b and significant correlation analyses -there is no significant difference in MEP latency as stated in the above paragraph.Why is this correlation relevant as there is no significant change?I think this might just confuse the reader and it doesn't really add to the story so I think you should delete it.
In the last sentence of your discussion you write "Importantly, the effects of PMd iTBS on the I-wave circuits were less in older compared to young adults."Although it is true your results support difference in older compared to younger adults, I don't believe a single analysis post-iTBS revealed a direct difference between young vs. old, so this is misleading.

Response to Reviewing Editor:
1. Please ensure that precise p values are stated throughout, rather than just > or <.
Precise P-values (to three significant figures) are stated in the manuscript, except where multiple comparisons are covered by a single statement or P-values are less than 0.001.Referee #1: 1. What, more specifically, does "low-level activation of the right FDI" refer to?

Response to
Rejection of trials with baseline EMG activity above 25 μV is mentioned later on, so was AMT recorded during contraction with maximum amplitudes of 25 μV?
Low-level activation refers to ~10% voluntary contraction of the right FDI.Participants were instructed to maintain ~10% contraction of the muscle during active TMS recordings (Hamada et al., 2013;D'Ostilio et al., 2016).This has now been stated in the manuscript (page 7).In addition, rejection of trials due to EMG activity above 25 µV was only completed for trials recorded in the resting muscle.This has now been clarified (page 9).

"Following AMT, the stimulus intensities producing a standard MEP amplitude approximating 1 mV (MEP 1mV
; PA 1mV and AP 1mV ), in addition to an MEP amplitude approximating 0.5 mV (MEP 0.5mV ; PA 0.5mV and AP 0.5mV ), when averaged over 20 trials, were identified" -What was the procedure for this?For example, was the stimulator intensity systematically varied after averaging 20 trials at each intensity?Was PEST used?
During these measures, we estimated an intensity and sampled 20 trials for each of the conditions.The intensity was adjusted and the trials were re-recorded if the average MEP amplitude did not fall in the range of 0.8-1.2mV for MEP 1mV and 0.4-0.6 mV for MEP 0.5mV .This process has now been stated in the manuscript (pages 7-8).

"For all measures of SICF, individual MEP amplitudes produced by paired-pulse
stimulation were expressed as a percentage of the mean MEP amplitude produced by single-pulse stimulation recorded at baseline (Cash et al., 2009;Liao et al., 2022) It has previously been shown that there is a correlation between the increase in single-pulse MEP amplitude and increase in SICF after I-wave periodicity TMS (iTMS), and that normalising to the post-iTMS MEP amplitude underestimates the change in excitability of the I-wave generating networks (Cash et al., 2009).To illustrate this point theoretically, if there is a two-fold increase in single-pulse MEP amplitude after an intervention (e.g. 1 to 2 mV), a two-fold increase in paired-pulse TMS (e.g. 2 to 4 mV) would result in no change in SICF after the intervention (when normalising to the post-intervention test MEP), even though there has been an absolute increase in SICF.Consequently, we also found a significant relationship between single-and paired-pulse MEP amplitude post-iTBS.Therefore, to avoid underestimating the effect of iTBS on I-wave facilitation, we have expressed post-iTBS SICF responses as a percentage of pre-iTBS single-pulse MEP amplitude, which is in line with previous studies (Cash et al., 2009;Opie et al., 2021;Liao et al., 2022).We have now explained this rationale and process in the manuscript (pages 9-10).Thank you to the reviewer for this suggestion.We have changed the correlation analysis to include FDR with Benjamini-Hochberg procedure.The details are now included in the methods and results section of the manuscript (pages 11, 15).

The use of the term "low-intensity" is confusing/misleading, I presume this refers to the use of peri-threshold intensities, but it could be interpreted as sub-threshold or as low intensity absolute values (for example <30% MSO).
We have removed the term "low-intensity" throughout the manuscript and directly referenced the measure as MEP 0.5mV .

What did participants do in the 1-hour intervals before and after iTBS and in the gap between 5 and 40 min post-iTBS measurements?
Participants remained seated and relaxed during breaks between TMS blocks as per instructions, but were allowed to read or use mobile devices using their left hand.Participants were specifically asked to maintain relaxation of the right hand during the experiment and this was monitored throughout.This is now stated in the manuscript (page 7).

"
We found that both PA SICF1.5 and AP SICF4.5 were potentiated (~20-40% increase) across both young and older adults.As the 1.5ms ISI assesses early I-waves and 4.5 ms ISI assesses late I-waves (Ziemann et al., 1998;Opie et al., 2018), one interpretation of these results could be that PA SICF 1.5 activates PA early I-waves, whereas AP SICF 4.5 activates AP late I-waves.This would be consistent with our single-pulse findings for PA 0.5mV and AP 0.5mV , and suggests that PMd iTBS may preferentially modulate both PA early and AP late I-waves."-Two points on this.First, I think the authors should remove the reference to "early PA" and late AP" throughout, as it implies that PA only activates early I waves, and AP only activates late I waves, in addition to suggesting that the late I waves activated by one orientation are different from the late I waves activated by another (ditto for early I waves).It would be better to simply refer to early or late I waves when naming the I waves.Second, it is later mentioned that "it is possible that the measure of SICF was complicated by the MEP1mV test stimulus, which likely resulted in mixed recruitment of I wave circuits (Opie et al., 2021)", which is a good point.Could the detection of an effect on SICF 4.5 using AP and on SICF 1.5 using PA reflect that these different orientations preferentially (but not solely) activate the I-waves that are intended to be interrogated by these different ISIs, and therefore there is more to be facilitated/inhibited.For example, if AP orientation engages late I wave circuits more so than PA orientation, then it seems logical that AP orientation would be a better orientation to use when attempting to facilitate the late I waves with SICF 4.5 , and would be more sensitive to effects of iTBS on said measure.
We agree with the reviewer's interpretation of our findings.We have therefore removed the explanation of early and late PA, and early and late AP I-waves in favour of early and late Iwaves, in addition to altering the discussion of SICF (pages 16-19).However, we felt that it was important to highlight the growing literature showing the possibility that PA and AP TMS can activate distinct early and late I-waves, and have briefly covered this point in the discussion of SICF (pages 17-18).

Response to Referee #2:
1.Although Figure 1 alludes to it, it is not clearly stated in text that the left PMd was always targeting.Please make this clear in the methods section for iTBS Targeting of left PMd has now been stated in the methods section for iTBS (pages 8-9).
2. In the data analysis section you state "as Spearman's correlation analysis revealed that post-intervention increases in single-pulse test MEP amplitudes predicted increases in the paired-pulse MEP amplitudes (ρ = 0.8, P < 0.05)".Are these results from your work or the previous work?If your work, please include them in the results section.
The result of this correlation analysis is now included in the results (page 14).

A visual from BrainSight for the stimulation location of PMd for all participants on standard MNI-ICBM152 template as well as a table with MNI coordinates (this could be supplemental figures and table). I think this is particularly important since PMd location was not based off function or structure from individualized MRIs.
Our PMd location was measured relative to M1 hotspot, and we recorded these locations in BrainSight in order to maintain consistent coil positioning when delivering PMd iTBS.We have clarified this in the methods section of the manuscript (page 9), in addition to including a visual and table of MNI coordinates of both M1 and PMd for all participants (See supplementary materials).

In the data analyses, you collapsed the MEP latency data across multiple stimulation intensities. Please provide data (even in supplemental data
) that there were no differences in MEP latency across PA1mV, AP 1mVn PA0.5mV and AP0.5mV.
We did not assess MEP latency from the single-pulse TMS data used for MEP amplitude analysis.Rather, our method for calculating MEP latency data follows the accepted practice of using separate recordings of 110% AMT PA for PA latency, 110% AMT AP for AP latency, and 150% of AMT LM for LM latency (Hamada et al., 2013).Together, the average latencies from these active muscle recordings were used to calculate PA-LM and AP-LM latencies (Hamada et al., 2013).We have clarified this process in the methods section for data analysis of the manuscript (page 8-9).

Why did the analyses investigating the changes in corticospinal excitability following the intervention using baseline-normalized
values not look at the PA 1mV , AP 1mV , PA 0.5mV and AP 0.5mV altogether?This is the statistical method you used for the SICF measures.
Our statistical method for both single-pulse and paired-pulse measures post-intervention were the same.Specifically, each individual measure (PA 1mV , AP 1mV , PA 0.5mV , AP 0.5mV , PA SICF 1.5 , PA SICF 4.5 , AP SICF 1.5 , and AP SICF 4.5 ) was examined using separate models (eight total) that investigated the factors of session (iTBS, sham), time (5 minutes, 40 minutes), and age (young, older).We have clarified this process in the methods section for statistical analysis of the manuscript (pages 10-11).We have corrected this section of statistical analysis to reflect Spearman's rank-order correlation analysis (page 11).

In
7. Due to the inability to record several of the measures, it would be important for the authors to include which participants (example participant code) and their demographic variables were excluded, and the remaining demographic variables for those left in the analyses (again, this is something that could be put in supplemental data).I believe it is interesting to the readers to understand what may have been different about these participants and why might that have contributed to not being able to record these measures.
We agree with the reviewer that there are demographic variables that may contribute to the difficulty of recording certain TMS measures.However, the demographic variables recorded in the present study (age, sex, and activation threshold) do not appear to show any meaningful contribution.We have therefore chosen to briefly highlight these details for the participants in whom we were unable to make TMS recordings in the results section of the manuscript (page 11).

I am curious why you also didn't include PA1mV over time in young vs. old within
Figure 2? I know there was not statistically significant difference, but it provides a nice visual contrast to the AP1mV already included in Figure 2 (part C).
Thank you to the reviewer for this suggestion.We have now added the graph for PA 1mV over time in young and older adults (Fig. 2C).
9. Older adults failed to demonstrate that they were different with iTBS compared to sham with PA 0.5mV but did demonstrate for AP 0.5mV .When taken into consideration with the older adults only demonstrating a significant increase in AP 1mV 40 (vs. 5) minutes post, what can that all mean?Are these changes correlated?
Thank you to the reviewer for highlighting this point.We have suggested that the absence of facilitation for PA 0.5mV in older adults indicates a weakened direct influence of PMd on early I-wave excitability.We also speculated that the time course delay in facilitation for AP 1mV in older adults may have been the result of an indirect mechanism, due to the weakened sensitivity in inhibitory circuits that received PMd projections, as previous work has shown that changes in excitatory (I-wave) circuits following PMd iTBS are preceded by changes in inhibitory circuits (Meng et al., 2020).Given that AP 1mV likely recruited more early I-waves compared to AP 0.5mV , this suggests that there may be both direct (absence of facilitation) and indirect effects (time course delay in facilitation) of ageing on PMd communication with early I-waves.We have now included this interpretation in the revised manuscript (pages 16-17).
10.No differences between young vs. older adults for SICF PA1.5mV increased at 40 minutes and compared to sham while AP SICF 4.5mV increased after iTBS compared to sham.AP SICF1.5 did not change but there was no discussion of PASICF 4.5mV results?Please include a statement regarding PA4.5mV SICF.
We have now included a discussion on both PA SICF 4.5 and AP SICF 1.5 (pages 17-18).
11.In regards to Figure 5b and significant correlation analyses -there is no significant difference in MEP latency as stated in the above paragraph.Why is this correlation relevant as there is no significant change?I think this might just confuse the reader and it doesn't really add to the story so I think you should delete it.
We agree with the reviewer and have removed Figure 5B and modified our discussion to better reflect this point (page 19).
12. In the last sentence of your discussion you write "Importantly, the effects of PMd iTBS on the I-wave circuits were less in older compared to young adults."Although it is true your results support difference in older compared to younger adults, I don't believe a single analysis post-iTBS revealed a direct difference between young vs. old, so this is misleading.
We have reworded this sentence to more accurately reflect our findings related to differences in iTBS effects for young and older adults (page 15).

12-Apr-2023 1st Revision -Editorial Decision
Dear Mr Liao, Re: JP-RP-2023-284204R1 "Modulation of dorsal premotor cortex differentially influences I-wave excitability in primary motor cortex of young and older adults" by Wei-Yeh Liao, George M. Opie, Ulf Ziemann, and John G. Semmler Thank you for submitting your manuscript to The Journal of Physiology.It has been assessed by a Reviewing Editor and by 2 expert referees and we are pleased to tell you that it is acceptable for publication following satisfactory revision.
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-All relevant 'n' values must be clearly stated in the main text, figures and tables, and the Statistical Summary Document (required upon revision).
-The most appropriate summary statistic (e.g.mean or median and standard deviation) must be used.Standard Error of the Mean (SEM) alone is not permitted.
-Exact p values must be stated.Authors must not use 'greater than' or 'less than'.Exact p values must be stated to three significant figures even when 'no statistical significance' is claimed.

22-Feb-2023
Thank you for re-submitting your work to the Journal of Physiology, which has been carefully reviewed by the original 2 expert reviewers and the editorial team.The reviewers are happy that you have adequately addressed their comments and have no remaining concerns.Although many of the statistical questions have been resolved, there are a few points that still need to be addressed.
In addition, thank you for making most of the p values exact.However, there are still some that are shown as < or >, and are not describing the results of multiple tests (e.g.In addition, baseline MEP latencies differed between coil orientations (F1,156 = 247.41,P < 0.05), with shorter PA-LM latencies compared to AP-LM latencies (EMD = 1.9 ms [1.6, 2.1], P < 0.05).).
p-values will need to be given precisely throughout before the paper can be accepted for publication.In addition, while it is good to see all the data points on the figures, the error bars are not described -I presume these are SD, but this is not stated.Please could the authors clarify for each figure.
Senior Editor: Please ensure that the basis of the calculation of errors bars is stated in all instances (and that this is bodied in standard deviations, unless there are grounds for showing confidence intervals).Please also see the note from the Reviewing Editor concerning the reporting of p values.

Response to Reviewing Editor:
1.In addition, thank you for making most of the p values exact.However, there are still some that are shown as < or >, and are not describing the results of multiple tests (e.g.In addition, baseline MEP latencies differed between coil orientations (F1,156 = 247.41,P < 0.05), with shorter PA-LM latencies compared to AP-LM latencies (EMD = 1.9 ms [1.6, 2.1], P < 0.05).).P-values will need to be given precisely throughout before the paper can be accepted for publication.
Precise P-values are stated in the manuscript to three significant figures.P-values smaller than 0.0001 are stated as P < 0.0001, whereas P-values are stated as < or > 0.05 where multiple comparisons are covered by a single statement.
2. In addition, while it is good to see all the data points on the figures, the error bars are not described -I presume these are SD, but this is not stated.Please could the authors clarify for each figure.
The data in the figures are presented as estimated marginal means (EMM) with 95% confidence intervals (95% CI), whereas the individual data points are the individual subject means.This has now been stated for each figure.

Response to Senior Editor:
1. Please ensure that the basis of the calculation of errors bars is stated in all instances (and that this is bodied in standard deviations, unless there are grounds for showing confidence intervals).Please also see the note from the Reviewing Editor concerning the reporting of P-values.
We have now stated mean ± standard deviation or EMM [95% CI] in all instances where appropriate throughout the manuscript and addressed the point from the Reviewing Editor regarding the reporting of P-values. 12-May-2023

13-Apr-2023 2nd Revision -Editorial Decision
Dear Dr Liao, Re: JP-RP-2023-284204R2 "Modulation of dorsal premotor cortex differentially influences I-wave excitability in primary motor cortex of young and older adults" by Wei-Yeh Liao, George M. Opie, Ulf Ziemann, and John G. Semmler We are pleased to tell you that your paper has been accepted for publication in The Journal of Physiology.
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EDITOR COMMENTS
Reviewing Editor: Thank you for addressing the remaining concerns in your resubmission.
2nd Confidential Review

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Please include an Abstract Figure file, as well as the figure legend text within the main article file.The Abstract Figure is a piece of artwork designed to give readers an immediate understanding of the research and should summarise the main conclusions.If possible, the image should be easily 'readable' from left to right or top to bottom.It should show the physiological relevance of the manuscript so readers can assess the importance and content of its findings.Abstract Figures should not merely recapitulate other figures in the manuscript.Please try to keep the diagram as simple as possible and without superfluous information that may distract from the main conclusion(s).Abstract Figures must be provided by authors no later than the revised manuscript stage and should be uploaded as a separate file during online submission labelled as File Type 'Abstract Figure'.Please ensure that you include the figure legend in the main article file.All Abstract Figures should be created using BioRender.Authors should use The Journal's premium BioRender account to export high-resolution images.Details on how to use and access the premium account are included as part of this email.
with the responses to my previous queries and the associated manuscript amendments.Referee #2:The authors have addressed my previous concerns and I have no further comments.I believe the revised manuscript is still of high quality and worth acceptance.
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