The Distinct Electrophysiological Mechanisms in the Cortico-Striatal Circuit of LID Rats

1. Summary

Thank you very much for taking the time to review this manuscript. We sincerely thank you for the valuable time and constructive suggestions. We have carefully addressed all the comments point-by-point and have revised the manuscript accordingly. All modifications are highlighted in red/yellow in the revised manuscript. Detailed responses are provided below.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes/Can be improved/Must be improved/Not applicable

We gave the corresponding response in the point-by-point response letter.

Is the research design appropriate?

Yes/Can be improved/Must be improved/Not applicable

We gave the corresponding response in the point-by-point response letter.

Are the methods adequately described?

Yes/Can be improved/Must be improved/Not applicable

We gave the corresponding response in the point-by-point response letter.

Are the results clearly presented?

Yes/Can be improved/Must be improved/Not applicable

Are the conclusions supported by the results?

Yes/Can be improved/Must be improved/Not applicable

We gave the corresponding response in the point-by-point response letter.

Are all figures and tables clear and well-presented?

Yes/Can be improved/Must be improved/Not applicable

We gave the corresponding response in the point-by-point response letter.

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Overinterpretation of “directionality” and causal language
The central claim of the manuscript is that M1 “drives” abnormal activity in the DLS and that this one-way influence may be a key cause of dyskinesia. However, cross-regional PAC alone does not establish causal directionality. The present data support an asymmetry in coupling (M1 phase / DLS amplitude) but not a definitive causal drive from M1 to DLS.

Please tone down the wording throughout the Abstract, Discussion, and Conclusions. Statements such as “M1 drives abnormal activity in DLS” or “one-way over-control” should be replaced by more cautious formulations. If the authors wish to support stronger directional claims, additional analyses would be needed (e.g., lag-based analyses, directed coherence, Granger-type approaches, or related methods).

Response 1: First, we sincerely thank the reviewer for raising this important and insightful concern. Our findings indicate that the observed PAC pattern (M1 phase / DLS amplitude) exhibits a distinct asymmetry, which may reflect an important pathophysiological feature of L-DOPA-induced dyskinesia (LID). We acknowledge that the present study was not designed to investigate the correlation or causal relationships between DLS and M1, such as through lag-based analyses, directed coherence measures, or Granger-causality approaches.

We fully agree that examining the directionality and causal interactions within the cortico-striatal network would provide valuable mechanistic insights into the development of LID. Accordingly, this question has become a major focus of our ongoing work. In future studies, we plan to systematically characterize DLS–M1 PAC dynamics at multiple time points across the 185-minute period following L-DOPA administration. In parallel, we will perform correlation, coherence, and causality analyses and integrate these findings with AIM scores to further elucidate the neural mechanisms underlying LID and the functional significance of asymmetric cross-regional PAC.

In response to the reviewer’s comment, we have also carefully revised the manuscript to avoid overinterpretation of PAC directionality. Specifically, the terminology throughout the Abstract, Introduction, Results, and Discussion sections has been consistently modified to “asymmetric cross-regional PAC.” In addition, we have added a dedicated Limitations section at the end of the manuscript to explicitly discuss the methodological constraints of PAC analysis and the inability of the current approach to infer causal relationships between brain regions.

We greatly appreciate the reviewer’s constructive suggestion, which has significantly improved the rigor and interpretation of our study. We thank the reviewer again for this valuable comment, which has helped us improve the clarity and scientific rigor of the manuscript. The corresponding revisions have been incorporated throughout the manuscript and are highlighted in the revised version.

The revised manuscript of these changes can be found : sections

‘Simply summary’- page 1, line 27

‘Abstract’-page 2, line 46

‘Introduction’-page 2, line 80-81

‘Results’ -page 11, line 394-397

‘Discussion’-page 12, line 431-434

Comments 2: Sample size, attrition, and unit-of-analysis are unclear
The manuscript initially states n = 15 animals per group, but different analyses use different group sizes (e.g., histology, LFP, spike analyses). It is therefore essential to explain how many animals were excluded at each stage and for what reason.
In addition, it is not sufficiently clear whether spike-based comparisons treat neurons as independent observations. If so, there is a risk of pseudoreplication because neurons are nested within animals.

Please provide: The number of animals initially assigned, lesioned successfully, implanted, excluded, and finally analyzed in each figure;explicit exclusion counts per group; a clear statement of the statistical unit for each analysis; and, ideally, a nested or mixed-effects approach for neuron-level analyses.

Response 2: We sincerely thank the reviewers for their valuable comments and suggestions. Below we provide our point-by-point responses. Initially, 15 rats were included in each group for the experiment. Both the PD and LID groups received intracranial 6-OHDA injections, and the success of PD model induction was confirmed in all animals via APO-induced rotation tests. Subsequently, the 15 rats in the LID group received intraperitoneal L-DOPA injections to induce the LID model, among which one rat was excluded due to an unqualified AIM score. Following electrode implantation in the three groups, varying degrees of loss occurred in each group. The specific allocation and exclusion details are presented in a table and provided as a supplemental table in the manuscript for full transparency.

The authors have also clearly indicated the number of animals included in each analysis in the figure legends of the Results section. Furthermore, the neuronal analyses in Figure 3 and Figure 6 have been re-analyzed at the animal level, replacing the previous neuron-level analyses to ensure more robust and conservative statistical inferences.

The revised manuscript of these changes can be found : sections

‘Materials and Methods’-page 5, line 204

‘Results’ -page 6, line 239\247\275\308\330\350\377\410

‘Appendix A Table A1’-page 17, line 604

Comments 3: Important information about bias control is missing
Random assignment is mentioned, which is appreciated. However, I could not find explicit information on: blinding during AIM scoring, blinding during histological assessment, blinding during spike sorting/classification, blinding during data analysis, or sample size/power calculation. Please clarify these points. If blinding was not performed, this should be acknowledged as a limitation.

Response 3: We thank the reviewer for raising this issue. As the reviewer correctly pointed out, the above analyses were indeed not performed in a blinded manner, which constitutes another limitation of this study. We greatly appreciate this suggestion, and in our future research, we will pay special attention to this issue and implement stricter data processing procedures.

The revised manuscript of these changes can be found : section

‘Limitation’-page 15, line 573

Comments 4: Exclusion criteria need to be defined more rigorously
The manuscript states that animals with incorrect electrode placements were excluded and that recordings without stable PAC peaks were excluded. These criteria need to be described in a more objective and transparent way.

Please specify: how incorrect placement was defined, how many animals/recordings were excluded for this reason in each group, how “stable coupling peaks” were defined, and how many recordings were excluded from PAC analyses in each group.

Response 4: We sincerely thank the reviewers for their valuable comments and suggestions. Below we provide our responses.

Regarding the electrode placement and data inclusion criteria, the expected electrode location for M1 was within cortical layers 3–5, and the depth for DLS was targeted within the range of 4.5–6.0 mm. Any recordings falling outside these ranges were considered electrode placement errors and were excluded from the analysis. Stable PAC coupling was defined as consistent peak intensity and location of coupling across different time points within the same recording session (e.g., per 10-s data segments over a 5-minute recording period). Data segments showing severe deviations in coupling intensity or range were excluded to ensure data quality. The specific details of animal exclusions, including the reasons for exclusion at each stage, are clearly presented in Appendix Table A1.

We thank the reviewer for this important comment. We agree that the original description of the exclusion criteria was insufficiently detailed. In the revised manuscript, we have clarified the criteria for both histological electrode-placement verification and PAC-related data inclusion, and we have added group-wise exclusion numbers in Appendix Table A1.

Incorrect electrode placement was defined based on post hoc Nissl-stained coronal sections. For M1 recordings, electrode tips/tracks were required to be located within layers III–V of the primary motor cortex. For DLS recordings, electrode tips were required to be located within the dorsolateral striatum, with the depth position falling within 4.5–6.0 mm. Animals were excluded if the electrode track or tip was outside the target region, could not be identified reliably, or was associated with substantial tissue damage that prevented anatomical verification.

We also revised the description of “stable coupling peaks” to avoid ambiguity and potential selection bias. In the revised analysis, PAC peak stability was defined as within-recording reproducibility of the dominant PAC pattern across short, non-overlapping time segments, rather than agreement with any expected frequency band, experimental group, or hypothesized direction of coupling. Specifically, each 5-min recording was divided into 10-s artifact-free epochs, and PAC comodulograms were computed independently for each epoch using the same frequency grid and surrogate-based significance threshold as in the main analysis. A recording was considered to contain a reliable PAC peak only when a suprathreshold PAC cluster was observed reproducibly across epochs and the centroid frequencies of the dominant clusters showed limited dispersion across epochs. This criterion was applied uniformly across all groups and before group-level statistical testing.

Importantly, this criterion was used only to ensure that centroid-based PAC measures, including centroid MI, phase frequency, and amplitude frequency, were derived from reproducible PAC structures rather than isolated noise-driven maxima. Recordings excluded from PAC-related analyses and the final numbers included in local PAC and cross-regional PAC analyses are now reported in Appendix Table A1. In the final dataset, local PAC analyses included 7 sham, 7 PD, and 6 LID animals, whereas cross-regional PAC analyses included 7 sham, 6 PD, and 6 LID animals.

The revised manuscript of these changes can be found : section

‘Materials and Methods’-page 5, line 189-198

Comments 5: Statistical strategy may be insufficient for the number of comparisons
The study involves multiple frequency bands, multiple brain regions, multiple neuronal subtypes, local and cross-regional PAC, and several quantitative endpoints. However, the statistical section only mentions one-way ANOVA with Tukey post hoc tests and unpaired t-tests.

Please clarify: whether assumptions of normality and homogeneity of variance were tested and whether primary outcomes were predefined. More detail is particularly important for the PAC analyses, where many frequency combinations are explored (multiple testing correction).

Response 5: We greatly appreciate the reviewer for raising this very valuable question for our future work. We acknowledge that normality and homogeneity of variance tests were not performed in the original manuscript. In the revised manuscript, we have now comprehensively addressed this issue. Specifically, we first applied the Shapiro–Wilk test to assess normality for all datasets, and Welch's test to assess homogeneity of variance. For data that did not follow a normal distribution, statistical comparisons were performed using the Mann–Whitney test (for two-group comparisons) or the Kruskal–Wallis test (for three-group comparisons). For data that followed a normal distribution, Welch's test was used to evaluate homogeneity of variance. Data violating the assumption of homogeneity of variance were analyzed using Welch's t-test or Welch's ANOVA with Dunn's multiple comparison test, whereas data meeting the assumption of homogeneity of variance were analyzed using unpaired t-test or ordinary ANOVA. All statistical methods have been updated accordingly in the revised manuscript.

The revised manuscript of these changes can be found : sections

‘Materials and Methods’-page 5, line 206-215

‘Results’

Comments 6: Conceptual issue: “pyramidal neurons” in the DLS

This is a major conceptual concern. In the striatum, it is not appropriate to refer to the principal projection neurons as “pyramidal neurons.” The Results themselves suggest that the relevant DLS populations are more consistent with fast-spiking interneurons and medium spiny neurons (MSNs).
Please revise the terminology throughout the manuscript. “Pyramidal neurons” should be reserved for cortical populations (e.g., M1), not for DLS neurons.

Moreover, internal methodological inconsistencies should be corrected. There appear to be at least two inconsistencies:

Response 6: We sincerely thank the reviewers for their valuable comments and suggestions. In the revised manuscript, all neurons recorded in the DLS have been uniformly reclassified as putative MSNs and putative projection neurons. This revision ensures more accurate and consistent nomenclature throughout the text, including the Abstract, Results, and Discussion sections.

The revised manuscript of these changes can be found : sections

‘Abstract’-page 1, line 39-40

‘Results’ -page 8, line 290

‘Discussion’-page 13, line 460-470

Comments 7: The apomorphine rotation test is described as a 15-minute recording, but successful lesioning is defined as >7 rotations per minute for at least 30 minutes. This is internally inconsistent and should be corrected.

Response 7: We sincerely thank the reviewers for their valuable comments and suggestions. We would like to clarify that during the experiment, the actual procedure involved recording the total number of rotations within a 15-minute period, and the criterion used for evaluation was >7 r/min. The authors have revised the manuscript accordingly to ensure the accuracy of the methodological description.

The revised manuscript of these changes can be found : section

‘Materials and Methods’-page 3, line 115-122

Comments 8: The selected electrophysiological analysis window is described as 80 min post-injection in Methods, whereas the Discussion refers to 85 min post-injection. Please reconcile this discrepancy.

Response 8: We sincerely thank the reviewers for their valuable comments and suggestions. We would like to clarify that the exact time point is 85 minutes, and the electrophysiological recording time point is consistent with the behavioral recording time. The authors have revised the manuscript accordingly to ensure accuracy and consistency in the methodological description.

The revised manuscript of these changes can be found : section

‘Materials and Methods’-page 4, line 159

Comments 9: The biomarker claim is too strong
The manuscript suggests that these findings may provide reliable biomarkers for LID. I think this is premature for a preclinical study of this type. It would be more appropriate to use terms such as “candidate electrophysiological signatures” or “potential biomarkers that require further validation.

Response 9: We thank the reviewer for suggesting a more rigorous and appropriate expression. The conclusion section of the manuscript has been revised to 'potential value as a biomarker for LID (section 4.6).

The revised manuscript of these changes can be found : section

‘Discussion’-page 15, line 553

Comments 10: There is a section numbering problem in the Results (duplicate section number 3.5).

Response 10: We thank the reviewer for the careful reading. This section (3.6) has been revised accordingly in the manuscript to address the point raised.

The revised manuscript of these changes can be found : section

‘Results’-page 9, line 337

Comments 11: Please carefully revise the manuscript for terminology consistency and English style.

Response 11: We thank the reviewer for the suggestion. The authors have carefully revised the manuscript and have made every effort to improve the English expression throughout the text.

The revised manuscript of these changes can be found : section

‘Discussion’-page 12, line 428-431

Comments 12: A data/code availability statement would significantly improve transparency and reproducibility.

Response 12: We agree that a clear data and code availability statement is important for improving the transparency and reproducibility of the study. Accordingly, we have added a “Data and Code Availability” statement to the revised manuscript. The statement clarifies that the data supporting the findings of this study, as well as the analysis code, are available from the corresponding author upon reasonable request, subject to institutional and ethical requirements.

The revised manuscript of these changes can be found : section

‘Data availability statement’-page 16, line 592-596

Comments 13: Only male rats were used; this should be stated more explicitly as a limitation.

Response 13: We sincerely thank the reviewers for their valuable comments and suggestions. This point has been addressed and explained in the newly added Limitations section, where we have clearly discussed the relevant methodological constraints.

The revised manuscript of these changes can be found : section

‘Limitation’-page 15, line 573-579

Comments 14: Cell-type classification based on extracellular waveform and firing properties should be justified more carefully, especially regarding the labels assigned to each subgroup.

Response 14: We sincerely thank the reviewers for their valuable comments and suggestions. We acknowledge that the confirmation of neuronal cell types indeed requires further investigation in subsequent studies. The classification in the present study was based on a comparison between our results and those reported in the literature. We greatly appreciate the reviewer's suggestion, and in our future research, we will design additional histological approaches to definitively identify the neuronal types.

The revised manuscript of these changes can be found : section

‘Limitation’-page 15, line 573-579

4. Response to Comments on the Quality of English Language

Point 1: The manuscript is understandable and generally readable, but the English would benefit from editing for:

-phrasing and grammar in several sections

-terminology consistency

-reduction of repetitive wording

-and clearer distinction between observation, interpretation, and speculation.

Response 1: We sincerely thank the reviewer for the detailed suggestions regarding the English writing. These comments will be of great help to the authors in their future manuscript preparation. The authors have carefully rechecked all the issues raised and have revised the relevant content accordingly throughout the manuscript.

1. Summary

Thank you very much for taking the time to review this manuscript. We sincerely thank you for ther valuable time and constructive suggestions. We have carefully addressed all the comments point-by-point and have revised the manuscript accordingly. All modifications are highlighted in red/yellow in the revised manuscript. Detailed responses are provided below.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes/Can be improved/Must be improved/Not applicable

We gave the corresponding response in the point-by-point response letter.

Is the research design appropriate?

Yes/Can be improved/Must be improved/Not applicable

We gave the corresponding response in the point-by-point response letter.

Are the methods adequately described?

Yes/Can be improved/Must be improved/Not applicable

We gave the corresponding response in the point-by-point response letter.

Are the results clearly presented?

Yes/Can be improved/Must be improved/Not applicable

We gave the corresponding response in the point-by-point response letter.

Are the conclusions supported by the results?

Yes/Can be improved/Must be improved/Not applicable

We gave the corresponding response in the point-by-point response letter.

Are all figures and tables clear and well-presented?

Yes/Can be improved/Must be improved/Not applicable

We gave the corresponding response in the point-by-point response letter.

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: In Figure 4, the authors suggest that enhanced PAC is associated with the dyskinetic state. However, no analysis was performed to examine the relationship between PAC strength and AIM scores. The authors should evaluate whether PAC correlates with dyskinesia severity to better support its potential relevance to LID.

Response 1: We thank the reviewer for raising this important question regarding behavioral relevance. In response, we added a supplementary animal-level AIM-PAC analysis using paired measurements collected before L-DOPA administration and at the 85-minute after-administration time point. We focused on the PAC centroid value because this metric was used in the main manuscript to characterize PAC coupling strength. The newly added Supplementary Figure A.3 shows the absolute AIM-PAC trajectories in selected LID rats. We observed that most animals exhibited increased PAC centroid values as AIM scores rose, particularly for M1 phase-to-DLS amplitude PAC and DLS local PAC. Nevertheless, both baseline and absolute PAC coupling strength varied substantially across individual animals. For this reason, we conservatively present these data as a descriptive supplementary observation, rather than as a formal AIM-PAC delta-correlation analysis. We have also revised the manuscript text to clarify this limitation. In future work, we plan to expand the sample size of animals and examine AIM-PAC relationships across multiple time points throughout the full L-DOPA response cycle.

The revised manuscript of these changes can be found : sections

‘Results’ -page 12, line 417-424

‘Appendix A’-page 18, line 612-620

Comments 2: In Figure 4 and Supplementary Figure A.1, PAC is present in the DLS but absent in M1, the inconsistence needs further explanation and discussion.

Response 2: We have conducted additional discussions on this section.(Section4.4)

Unlike the DLS, no significant enhancement of PAC was detected in the M1 region. This regional specificity suggests that LID-associated abnormal cross-frequency coupling may not be broadly distributed throughout the motor network, but instead may predominantly arise within local striatal microcircuits. As the primary target of dopaminergic replacement therapy, the striatum undergoes extensive changes in neuronal excitability and synaptic plasticity during the development of LID. Combined with the significant alterations in the firing properties of MSNs and FSIs observed in the present study, we speculate that aberrant dopaminergic stimulation disrupts the balance between excitation and inhibition within local striatal circuits, thereby promoting pathological synchronization of neuronal populations and enhancing the modulation of gamma-band activity by low-frequency oscillations. Consequently, the enhanced PAC observed in the DLS may reflect pathological network remodeling underlying abnormal motor information integration and output within the striatum. In contrast, although oscillatory power was also altered in the M1 region, these changes were not accompanied by a significant increase in local PAC. This finding suggests that cortical oscillatory abnormalities may primarily reflect alterations in neuronal activity levels or interregional synchronization rather than a reorganization of local cross-frequency coupling mechanisms. Given the complex laminar architecture of M1, its heterogeneous neuronal composition, and its extensive cortical and subcortical inputs, neuronal activity in this region is likely shaped by multiple converging information streams, which may reduce the likelihood of generating stable local PAC. Therefore, the role of M1 in LID may be more closely related to the integration and transmission of aberrant motor signals, whereas the generation and amplification of pathological cross-frequency coupling appear to occur predominantly within local striatal networks. Collectively, these findings further support a central role for striatal microcircuit remodeling in the pathophysiology of L-DOPA-induced dyskinesia.

The revised manuscript of these changes can be found : section

‘Discussion’ -page 14, line 497-523

Comments 3: In Figure 7, the author gets the conclusion that cortical activity drives striatal activity in LID with enhanced M1-to-DLS PAC. However, PAC analysis reflects coupling effect instead of cause effect, so it does not directly establish the direction of information flow. The authors should discuss the limitations of concluding directional interactions from PAC measurements alone.

Response 3: First, we appreciate you raising this critical question. Currently, the PAC (M1 phase/DLS amplitude) coupling only shows asymmetry, which may be a key etiological factor of LID disease; however, this study did not investigate the correlation or causality between DLS and M1 (e.g., through lag effect analysis, directional consistency tests, or Granger causality methods). We have added relevant content on this limitation in the "Limitations" chapter. Our future research will specifically examine the PAC between DLS and M1 at different time points within 185 minutes after L-DOPA administration, while conducting correlation, consistency, and causality analyses. Combined with AIM scores, we will further elucidate the pathological mechanisms of LID disease.

The revised manuscript of these changes can be found : sections

‘Simply summary’- page 1, line 27

‘Abstract’-page 2, line 46

‘Introduction’-page 2, line 80-81

‘Results’ -page 11, line 394-397

‘Discussion’-page 12, line 431-434

1. Summary

Thank you very much for taking the time to review this manuscript. We sincerely thank you for ther valuable time and constructive suggestions. We have carefully addressed all the comments point-by-point and have revised the manuscript accordingly. All modifications are highlighted in red/yellow in the revised manuscript. Detailed responses are provided below.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes/Can be improved/Must be improved/Not applicable

Is the research design appropriate?

Yes/Can be improved/Must be improved/Not applicable

Are the methods adequately described?

Yes/Can be improved/Must be improved/Not applicable

Are the results clearly presented?

Yes/Can be improved/Must be improved/Not applicable

Are the conclusions supported by the results?

Yes/Can be improved/Must be improved/Not applicable

We gave the corresponding response in the point-by-point response letter.

Are all figures and tables clear and well-presented?

Yes/Can be improved/Must be improved/Not applicable

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: The Abstract states that the LID group showed more FSIs but lower firing rates, whereas the Results primarily describe reduced firing rates and changes in subtype counts derived from spike classification. The presentation should be harmonized and clarify whether absolute numbers or recording proportions are being discussed.

Response 1: In the abstract of our manuscript, we aim to convey that compared with the sham group, the LID group exhibited a greater absolute number of recorded FSIs, although the firing rates of these neurons were reduced. The language describing this finding has been revised accordingly in the manuscript. Notably, we are referring here to the absolute numbers of recorded neurons, and the actual neuron counts for each animal group have been presented in the Results section.

The revised manuscript of these changes can be found : sections

‘Abstract’-page 1, line 39

‘Results’ -page 8 , line 284-294

Comments 2: The abbreviation table defines PAC as “Phase frequency coupling”, whereas throughout the manuscript PAC is correctly described as “phase–amplitude coupling.” This should be corrected for consistency.

Response 2: We would like to note that PAC stands for phase–amplitude coupling, and this abbreviation has been consistently corrected in the revised manuscript.

The revised manuscript of these changes can be found : section

‘Abbreviations’-page 16, line 602

Comments 3: Levodopa-induced dyskinesia (LID) is a severe motor complication of long-term levodopa (L-DOPA) in Parkinson's disease (PD), yet its underlying mechanisms underlying LID remain unclear and candidate biomarkers vary.

Response 3 : Levodopa-induced dyskinesia (LID) is a severe motor complication associated with long-term levodopa (L-DOPA) treatment for Parkinson's disease (PD). Its underlying mechanisms remain unclear, and candidate biomarkers lack consistency.

The revised manuscript of these changes can be found : section

‘Abstract’-page 1, line 33-35

Comments 4: Neuronal subtype classification is based on extracellular waveform characteristics and firing properties and therefore should be interpreted as putative rather than definitive. The manuscript would benefit from a brief acknowledgment of this limitation and discussion of potential misclassification.

Response 4 : As suggested, we have reclassified all DLS neurons in the revised manuscript as putative MSNs and putative projection neurons. We recognize that conclusive cell-type identification will require further studies, and our current assignment is based on the electrophysiological features observed in our data and comparisons with prior reports. We are grateful for this valuable input and intend to pursue histological validation in future work. This limitation has also been acknowledged in the Limitations section

The revised manuscript of these changes can be found : sections

‘Abstract’-page 1, line 39-40

‘Results’ -page 8, line 290

‘Discussion’-page 13, line 460-470

‘Limitation’-page 15, line 573-579

Comments 5: The PAC analyses are interesting and potentially important; however, the manuscript occasionally overinterprets these findings. Cross-regional PAC demonstrates statistical coupling between oscillatory components but does not, by itself, establish causal information flow or directional connectivity. Statements referring to "direct evidence" of information transfer or cortical dominance should be tempered or supported with complementary directional analyses.

Response 5: We sincerely thank the reviewer for raising this very important point. In our current data, the PAC (M1 phase / DLS amplitude) coupling appears to be asymmetric, which may be a key feature of LID pathology. While we have not conducted directional or causal analyses between DLS and M1 in this study—such as lag-based analysis, directed coherence, or Granger causality—we have planned future experiments to examine PAC at various time points within 185 min post-L-DOPA injection. These experiments will combine correlation, coherence, and causality assessments, as well as AIM scores, to help us better understand the mechanisms underlying LID.

Accordingly, we have modified the wording regarding PAC directionality throughout the Abstract, Introduction, Results, and Discussion, and now use "asymmetric cross-regional PAC." We have also added a Limitations section at the end of the paper to explicitly discuss the constraints of our current PAC approach.

The revised manuscript of these changes can be found : sections

‘Simply summary’- page 1, line 27

‘Abstract’-page 2, line 46

‘Introduction’-page 2, line 80-81

‘Results’ -page 11, line 394-397

‘Discussion’-page 12, line 431-434

4. Response to Comments on the Quality of English Language

Point 1: The English could be improved to more clearly express the research.

Response 1: We sincerely thank the reviewer for the detailed suggestions regarding the English writing. These comments will be of great help to the authors in their future manuscript preparation. The authors have carefully rechecked all the issues raised and have revised the relevant content accordingly throughout the manuscript. 

1. Summary

Thank you very much for taking the time to review this manuscript. We sincerely thank you for ther valuable time and constructive suggestions. We have carefully addressed all the comments point-by-point and have revised the manuscript accordingly. All modifications are highlighted in red/yellow in the revised manuscript. Detailed responses are provided below.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes/Can be improved/Must be improved/Not applicable

Is the research design appropriate?

Yes/Can be improved/Must be improved/Not applicable

Are the methods adequately described?

Yes/Can be improved/Must be improved/Not applicable

We gave the corresponding response in the point-by-point response letter.

Are the results clearly presented?

Yes/Can be improved/Must be improved/Not applicable

Are the conclusions supported by the results?

Yes/Can be improved/Must be improved/Not applicable

Are all figures and tables clear and well-presented?

Yes/Can be improved/Must be improved/Not applicable

We gave the corresponding response in the point-by-point response letter.

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: A more detailed description of the surgical procedures is needed, including an indication of the type of anesthesia used. The authors used sodium pentobarbital for perfusion (line 134), but do not specify the type of anesthesia for 6-OHDA administration (line 105) or for electrode implantation (line 127).

Response 1: We have now supplemented the Methods section with detailed information regarding the surgical procedures and the type of anesthesia used. All animals were anesthetized via intraperitoneal injection of sodium pentobarbital, and we have clearly indicated that the dosages administered for surgical anesthesia and for transcardial perfusion were different.

The revised manuscript of these changes can be found : section

‘Materials and Methods’-page 3, line 105-122\133-142

Comments 2: Section 3.4 (lines 247-252). Are data from the control group presented in this paragraph? This requires clarification. This clarification is also needed in the legend to Figure 3B (lines 266-267) and in the legend to Figure 6B (line 332).

Response 2: We would like to clarify that the data presented in both Figure 3B and Figure 6B are derived from the same sham group. This clarification has been clearly stated in the revised manuscript to avoid any potential confusion.

The revised manuscript of these changes can be found : section

‘Results’ -page 9, line 311; page 11, line 379

Comments 3: In the figures and figure legends, the authors sometimes refer to the control group of rats as "CON," sometimes as "sham," and sometimes (Figure 5) as "control(sham)" or "sham controls." Often, one designation is used in the figure, and another in the figure legend. Consistency is needed.

Response 3: Following the reviewer's suggestion, we have consistently applied the term "sham" to refer to the control group throughout the entire manuscript.

The revised manuscript of these changes can be found : sections

‘Materials and Methods’-page 3, line 105

‘Results’ -page 5-12

Response to Reviewer 1 Comments

1. Summary

Thank you very much for taking the time to review the new manuscript. We sincerely thank you for the valuable suggestions. We have carefully addressed all the comments and have revised the manuscript accordingly. All modifications are highlighted in red/yellow in the new revised manuscript. Detailed responses are provided below.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes/Can be improved/Must be improved/Not applicable

We gave the corresponding response in the point-by-point response letter.

Is the research design appropriate?

Yes/Can be improved/Must be improved/Not applicable

We gave the corresponding response in the point-by-point response letter.

Are the methods adequately described?

Yes/Can be improved/Must be improved/Not applicable

We gave the corresponding response in the point-by-point response letter.

Are the results clearly presented?

Yes/Can be improved/Must be improved/Not applicable

Are the conclusions supported by the results?

Yes/Can be improved/Must be improved/Not applicable

We gave the corresponding response in the point-by-point response letter.

Are all figures and tables clear and well-presented?

Yes/Can be improved/Must be improved/Not applicable

We gave the corresponding response in the point-by-point response letter.

3. Point-by-point response to Comments and Suggestions for Authors

Comments and Suggestions for Authors
The revised manuscript is substantially improved and addresses several of the main concerns raised in the previous round. However, the main issue regarding overinterpretation of cross-regional PAC directionality has been only partially resolved. Although the revised version now uses more cautious wording in some places, several statements still remain stronger than warranted by the data. In particular, the Simple Summary still suggests that the asymmetric coupling pattern “may be a key cause of dyskinesia,” and the Discussion still refers to the findings as direct evidence of abnormal functional connectivity in a way that may overstate what cross-regional PAC can support. The data support an asymmetric coupling pattern, but not a definitive causal or directional drive from M1 to DLS. This should be toned down consistently throughout the manuscript. In addition, although the statistical section has been expanded, some aspects remain unclear or incomplete. The revised manuscript still does not fully address whether primary outcomes were predefined, and the issue of multiple comparisons, particularly relevant for PAC-related analyses, remains insufficiently discussed. I also could not identify a clear statement regarding sample size or power calculation.

Response 1: We sincerely thank the reviewer for this valuable suggestion. We fully agree that cross-regional PAC reflects statistical coupling rather than causal or directional interactions. Accordingly, we have carefully revised the manuscript to ensure that all interpretations are strictly consistent with the evidence supported by the data. Specifically, we have replaced all statements implying causal influence or directional drive with more cautious descriptions, such as “asymmetric cross-regional coupling” or “altered interregional coordination,” throughout the Simple Summary, Introduction, Discussion, and Conclusions. In addition, we have further expanded the Limitations section to explicitly state that cross-regional PAC alone cannot determine the directionality or causality of cortico-striatal interactions. We also emphasize that future studies incorporating directional connectivity analyses (e.g., Granger causality, directed coherence, or lag-based approaches) will be necessary to further elucidate this issue.

The new revised manuscript of these changes can be found : sections

‘Simply summary’- page 1, line 27-30

‘Introduction’ - page 3, line 87-89

‘Discussion’- page 18, line 531-533

- page 19, line 568-572

- page 20, line 589-591/592-594

‘Conclusion’- page 21,line 608-610

Response 2: We sincerely thank the reviewer for the further valuable suggestions regarding the statistical analysis. In response, we have clarified the Statistical analysis section by explicitly stating that the primary outcome measures focused on PAC modulation index and centroid value. We have also clarified that statistical comparisons were performed on summary PAC metrics rather than individual frequency bins, thereby avoiding extensive multiple-comparison testing across the full phase–amplitude coupling comodulogram. In addition, we have added a statement indicating that no formal a priori sample size calculation was performed. The sample sizes were determined based on previous electrophysiological studies using similar experimental paradigms, as well as ethical considerations aimed at minimizing the use of experimental animals. These revisions have been incorporated into the Methods and Limitations sections to improve the transparency, rigor, and reproducibility of the study.

The new revised manuscript of these changes can be found : sections

‘Materials and Methods’- page 7, line 224-227

- page 7, line 230-233

- page 7, line 244-246

‘Limitation’- page 21,line 626-630

4. Response to Comments on the Quality of English Language

Comments on the Quality of English Language

The revised manuscript is substantially improved and generally understandable, but it would still benefit from one final round of careful English editing before acceptance. The remaining issues are mostly stylistic and linguistic rather than conceptual. For example, some expressions remain slightly awkward or non-idiomatic, such as “while no clear coupling was observed in inverted manner,” “used by Welch’s ANOVA,” or “fallowed by Dunn’s multiple comparison test,” and a few neuronal terms are still inconsistently phrased (e.g., “medial spiny neurons” instead of “medium spiny neurons”). In addition, some sentences in the Results and Statistical Analysis sections are grammatically correct only in part but remain difficult to follow because of word order or phrasing, and certain descriptions of the PAC findings could still be softened slightly to better distinguish observation from interpretation.

Response 1: In response to the reviewer’s suggestion, the manuscript has undergone a comprehensive English language revision and stylistic refinement to improve clarity, readability, and consistency. All sections have been carefully reviewed to ensure that the language meets the standards of scientific publication.

The revised manuscript of these changes can be found in revised maniscript.

Sections: ‘Simply summary’; ‘Abstract’; ‘Introduction’ ;‘Materials and Methods’; ‘Results’; ‘Discussions’; ‘Conclusions’; ‘Limitations’.