Alterations in the intrinsic properties of striatal cholinergic interneurons after dopamine lesion and chronic L-DOPA

Acceptance summary:

Prior work showed that striatal cholinergic interneurons are required for levodopa-induced dyskinesia, but it was unclear whether and how cholinergic interneuron properties changed in these conditions. Using ex vivo electrophysiology, this paper provides convincing evidence that cholinergic interneurons indeed show changes in their spontaneous firing rates, and furthermore identified that both HCN and SK ionic currents are involved in the underlying cellular mechanisms. This cellular mechanism of both translational and basic science interest, and provides important insights into the understanding of levodopa-induced dyskinesia.

Decision letter after peer review:

Thank you for submitting your article "Alterations in the intrinsic properties of striatal cholinergic interneurons after dopamine lesion and chronic L-DOPA" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Jun Ding as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by John Huguenard as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Joshua A Goldberg (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.

The reviewers are impressed by the thorough analysis of the properties of striatal cholinergic interneurons (ChI) in parkinsonian animals chronically treated with levodopa. The topic is of both translational and basic science interest. In addition, the reviewers think that this will be one of the highest quality studies in ChI in the literature, once published. The physiology is of a very high quality, the writing is exceptionally clear, and the overall conclusions are well-justified.

The reviewers did raise some concerns. However, very minor additional experiments have been requested. The main revision should focus on clarity and presentation, and critically on data reporting.

1) Data reporting: please report both number of cells and number of animals in each figure.

2) Data reporting: for readability, it would be useful to put numbers for some of the major findings in the Results text, not only in the figures/figure legends, mean =/- SEM. For example, the average firing rate of ChI and the n for the experiment.

3) The experiment described in Supplementary Figure 3 is a bit confusing. Please clarify and rationale and conclusion from the D1 and D2 antagonist experiments.

4) In some data sets, the number of replicates is small, for example, n=3-8. Even though the conclusion is clear, the reviewers think that the minimum n number for such electrophysiology experiments should be n=6-8.

5) Please add some discussion of whether L-DOPA treatment alone (in healthy animals) could potentially affect ChI physiology.

6) Please add discussion on the potential relationship between change of ChI dendritic morphology and physiological findings.

Reviewer #1:

It has been shown for decades that cholinergic modulation mediated by striatal cholinergic interneuron (ChI) and activity of ChI itself are involved in Parkinson's disease (PD) and L-dopa induced dyskinesia (LID). However, the basis of changes in ChI physiology in PD and LID is still not clear. In the manuscript by Choi and colleagues, the authors performed thorough electrophysiological analysis on the firing of ChI in acute brain slices from control, DA depleted and L-dopa treated mice. They identified that both HCN and SK currents were decreased in the DA depleted condition, in addition, HCN currents were rescued after L-dopa treatment, but SK current remained depressed after chronic L-dopa. These changes of intrinsic properties can explain the decreased firing frequency seen in DA depleted state, and elevated firing frequency in L-dopa treated condition. To confirm the changes in HCN and SK, the authors performed thorough pharmacological manipulations and analysis, as well as the HCN and SK mRNA expression levels. It would be fantastic to directly manipulate ion channel activity selectively in ChIs in vivo and examine the behavioral consequences. However, such manipulation (especially fine adjustment of ion channel activity to certain level in vivo) is technically not possible. Overall, the data quality is high. It would be one of the most thorough analysis of ChI physiology in DA depletion and L-dopa treatment conditions. The overall conclusion is well supported by the experimental data. I do have a couple of concerns/suggestions:

1) What is the effect of chronic L-dopa treatment on ChI physiology? This would be valuable to access the increased firing is caused by L-dopa treatment or requires prior DA depletion.

2) What is the relationship between the change of ChI dendritic arborization and HCN, and/or SK current? This is particularly relevant because HCN is highly expressed in dendrites. More discussion would be helpful.

3) The statistical and data reporting can be further improved.

4) The data included in Supplementary Figure 3 are particularly confusing. It is not clear what the authors want to tell here. It seems that either D1 or D2 antagonist alone could significantly decrease the firing frequency in DA depleted and LID conditions. However, the statistical analysis here is very confusing.

Reviewer #2:

In this manuscript, Choi et al. examine the properties of striatal cholinergic interneurons (ChI) in parkinsonian animals chronically treated with levodopa. Prior work showed that ChI are required for levodopa-induced dyskinesia (LID), but it was unclear whether and how ChI properties changed in these conditions. Using ex vivo electrophysiology, the authors find that ChI indeed show changes in their spontaneous firing rates, and furthermore are able to identify some of the underlying cellular mechanisms. The topic is of both translational and basic science interest, and this will be one of the highest quality studies in ChI in the literature, once published. The physiology is of a very high quality, the writing is exceptionally clear, and the overall conclusions are well-justified. However, I have a few concerns:

1) The authors have three main conditions: healthy control, 6-OHDA (dopamine depleted), and 6-OHDA (dopamine depleted) + levodopa treated. Their key finding is that the firing rate of ChI is reduced in the 6-OHDA condition, but restored to above healthy control levels in the 6-OHDA + levodopa condition. Ideally, they would have included a fourth group, healthy control + levodopa, to assay whether levodopa treatment can cause changes in healthy ChI as well, or whether there is a synergy between the parkinsonian striatal changes and the levodopa. If they are unable to include/add this fourth group, it would be valuable to comment on what the authors think might be going on in this condition.

2) The critical findings of this paper are related to the intrinsic properties of ChI, and the numbers of cells recorded are quite large for these primary analyses. However, they have also included data related to ChI synapses (sIPSC, sEPSCs), which have much smaller sample sizes, and indeed, one of their experiments (sEPSCs) shows no difference between conditions. Given the typical variability in sEPSC and sIPSC or mEPSC and mIPSC measurements, from slice to slice, mouse to mouse, and cell to cell, I think a larger n is required to definitively say whether ChI synapses are similar or different across conditions. I would favor increasing the n, dropping this section, or rephrasing the text to acknowledge these experiments are underpowered.

3) At the end of the Results section, the authors include data where they have washed on dopamine. While potentially interesting, the relationship of these experiments to their overall result is not clearly articulated, and again, the n is exceptionally small (3-8) by comparison to the robust numbers for their central findings.

Reviewer #3:

The manuscript of Choi and co-workers addresses the issue of alterations in the firing rate of striatal cholinergic interneurons (ChIs) upon treatment with 6-OHDA, which causes hemiparkinsonism, and then following chronic levodopa induced dyskinesia. While there is clear evidence for decades that dopamine depletion leads to adaptation in cholinergic signaling in the striatum, it has been difficult to determine what actually happens to ChIs which are the main source of tonic striatal acetylcholine (ACh). Several papers have addressed this issue and have reach contradictory results. I myself have tried this for decades while at various labs including my own and have not been able to reach a clear finding. Dr. Kang is one of the first to have reported a clear increase in the firing rate of ChIs after induction of LID in a seminal paper published a decade ago. This finding has been replicated since by several groups, but whether there is a change after 6-OHDA treatment has been hard to tell.

In this manuscript, the authors put this question to rest with incontrovertible evidence that there is indeed a reduction in the firing rate of ChIs. But more importantly, the authors have provided compelling mechanistic evidence that the HCN and SK currents are responsible for these changes in firing rate. They have done this with the use of direct measurements of these currents showing that they are altered with the various treatments. They also showed that pharmacological manipulation of these currents can phenocopy the effect. It would have been nice to have seen a rescue experiment, however, this is not really possible because we don't have reliable drugs to boost HCN or SK currents. They have also measured the transcript of these channels subunits. So, as far as I'm concerned, they have done an excellent job.

In addition to the high technical quality and commendable rigor of the work, and the resolution of this long standing question, there is great clinical relevance to this finding. It has been known for decades that the anticholinergic therapy can be effective to treat parkinsonism, albeit with unpleasant side effects. Discovering the precise biophysical mechanism of the adaptation in ChI firing rates, provides new potential therapeutic targets. For these reasons, I find this manuscript, highly relevant to the general audience of neuroscientists and biologists.

I think this study is well done and basically ready.