Optogenetic Inhibition of Striatal Parvalbuminergic Interneurons Unmasks Impaired GABA and Adenosine Signaling in DYT1 Knock-In Mice

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this study, the authors combined optogenetic inhibition of parvalbumin-positive fast-spiking interneurons (PV+ FSI) with in vivo microdialysis to investigate the striatal neurotransmitter dynamics in a mouse model of DYT1 dystonia. Particularly, they evaluated the extracellular levels of several neurotransmitters, revealing genotype-specific differences regarding GABA and adenosine signaling in response to PV+FSI optoinhibition. However, I have some suggestions for the author before proceeding with the publication.

1. Line 91. The sentence does not correspond to any table.
2. Figure 2-7. The authors could represent the bar graphs (a) and single value (b) together in a single graph. This could be applied for all figures.
3. Line 154. The statistics are incomplete. 
4. Line 164. There is a typo in the title (PF instead of PV).
5. Results 2.2.5. The number of Ach’s samples could be increased to reach the number of animals required by power analysis.
6. Moreover, given that this syndrome is associated with movement disorders, the author could provide a behavioral correlate associated with the observed neurochemical alterations. Are these alterations associated with changes in synaptic plasticity? Last, did the authors observe sex differences?

Author Response

We sincerely thank the reviewer for the careful evaluation of our manuscript and for the constructive comments and suggestions. We appreciate the positive assessment of the introduction and methodological description. We have carefully revised the manuscript accordingly and addressed all comments point-by-point below. All changes made in the revised manuscript are highlighted accordingly.

1. Line 91. The sentence does not correspond to any table.

We thank the reviewer for noticing this inconsistency. The corresponding reference was corrected in the revised manuscript. (See Line 91)

2. Figure 2–7. The authors could represent the bar graphs (a) and single value (b) together in a single graph.

We thank the reviewer for this helpful suggestion. The figures were revised accordingly by integrating the group data and individual data points into single graphs to improve clarity and readability of the results.

3. Line 154. The statistics are incomplete 

F (1/53) =     , p < 0.05]

We thank the reviewer for identifying this issue. The missing statistical values were added and the corresponding section was corrected in the revised manuscript. (See Line 125)


4. Line 164. There is a typo in the title (PF instead of PV).

We thank the reviewer for pointing out this typo. The abbreviation was corrected from “PF” to “PV” in the revised manuscript. (See Line 135)


5. Results 2.2.5. The number of ACh samples could be increased to reach the number of animals required by power analysis.

We thank the reviewer for this valuable suggestion. We agree that increasing the number of  ACh samples would strengthen the statistical power of the cholinergic analyses. However, whether ACh concentrations exceeded the limit of quantification only became apparent after completion of the in vivo optodialysis experiments and subsequent LC-MS/MS analysis. Therefore, it was not possible to selectively increase the number of samples during the experimental procedure.

Nevertheless, we agree that future studies specifically optimized for cholinergic measurements, including extended sampling strategies or targeted analytical approaches, may help to increase the number of quantifiable ACh samples and further improve statistical robustness. (See Line 312 - 315)

 

6. Moreover, given that this syndrome is associated with movement disorders, the author could provide a behavioral correlate associated with the observed neurochemical alterations. Are these alterations associated with changes in synaptic plasticity? Last, did the authors observe sex differences?

We thank the reviewer for this important comment. The primary aim of the present study was to characterize extracellular neurotransmitter dynamics following optogenetic inhibition of PV+ FSI using the optodialysis approach. We agree, however, that the observed neurochemical alterations should be discussed in the context of their potential functional and behavioral relevance.

Therefore, we expanded the Discussion section to further address how the impaired GABAergic and adenosinergic responses in DYT1 KI mice may contribute to abnormal striatal network activity, impaired inhibitory control, maladaptive synaptic plasticity, and ultimately altered motor circuit function associated with dystonia. (See Line 232 - 237)

Behavioral effects of PV+ FSI inhibition in this mouse model were previously investigated in our earlier study (Schulz et al., 2023), where no overt dystonic symptoms were observed despite genotype-specific neuronal activity changes. This aspect has now been clarified in the revised manuscript. (See Line 376 - 383).

Both sexes were included with approximately equal distribution (See Supplementary Table-3). However, we did not perform sex-disaggregated statsitical analyses, as the study was not powered for such comparisons. Research into sex as a biological variable requires dedicated, sufficiently powered study designs (Shansky, R.M., Murphy, A.Z. Considering sex as a biological variable will require a global shift in science culture. Nat Neurosci 24, 457–464 (2021). https://doi.org/10.1038/s41593-021-00806-8). This was not the focus of the present study. 

 

We thank the reviewer for his insightful comments and suggestions, and hope that the changes in the revised manuscript are accordingly.

 

Reviewer 2 Report

Comments and Suggestions for Authors

This article investigates dystonia using the DYT1 KI mouse model. To elucidate the impact of PV+ FSIs on striatal neurotransmitter dynamics, the authors employed optodialysis—a combination of in vivo microdialysis and optogenetics—to modulate neuronal activity and monitor extracellular neurotransmitters in freely moving mice. Dialysates were collected during baseline (light off), stimulation (light on), and post-stimulation (light off) periods. This approach enabled the authors to simultaneously measure striatal extracellular levels of GABA, dopamine, 3-methoxytyramine, acetylcholine, iso-acetylcholine, choline, adenosine, and 5-hydroxyindoleacetic acid across baseline, light stimulation, and post-stimulation periods.

The authors found no significant differences between WT and DYT1 KI mice in basal extracellular concentrations of GABA, dopamine, or adenosine. In WT mice, optogenetic inhibition reduced GABA and adenosine levels. DYT1 KI mice showed no change in GABA and only a delayed reduction in adenosine during the post-stimulation period. Dopamine, choline, and 5-hydroxyindoleacetic acid were unaffected by optogenetic inhibition, except for a reduction of 5-hydroxyindoleacetic acid in the post-stimulation period in DYT1 KI mice. These results reveal genotype-specific differences in the response to optogenetic inhibition between DYT1 KI and WT mice, suggesting altered inhibitory control and neuromodulatory imbalance in DYT1 KI mice.

In the discussion, the authors thoroughly interpret their findings and highlight the advantages and limitations of the optodialysis approach. They conclude that optodialysis is a viable method for circuit-specific neurochemical profiling in vivo, and that the identified methodological constraints provide a clear roadmap for technical refinement in future studies.

The manuscript is accompanied by figures that clearly explain the approach used, which greatly facilitates understanding and strengthens the work. The supplementary materials include all raw data collected during the experiments, which also inspires confidence in the results. Overall, I found the work highly commendable; it is conducted at a high technological level with adherence to all ethical standards. While reading the article, I did not encounter any significant criticisms, and I believe the manuscript should be accepted for publication in IJMS.

Author Response

We would like to express our gratitude to the reviewer for their very positive assessment of our work. We are very pleased that the reviewer found our study to be technically sound, ethically conducted, and worthy of publication without major criticisms. Their kind words are deeply appreciated.