Cross-pathway integration of cAMP signals through cGMP and calcium-regulated phosphodiesterases in mouse striatal cholinergic interneurons

  1. Ségolène Bompierre
  2. Yelyzaveta Byelyayeva
  3. Elia Mota
  4. Marion Lefevre
  5. Anna Pumo
  6. Jan Kehler
  7. Liliana R.V. Castro
  8. Pierre Vincent

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Abstract

Acetylcholine plays a key role in striatal function, yet the intricate dynamics of cyclic nucleotide signaling which govern the firing properties of cholinergic interneurons (ChINs) have remained elusive. Since phosphodiesterases determine the dynamics of cyclic nucleotides, in this study, we used FRET biosensors and pharmacological compounds to examine phosphodiesterase activity in ChINs in mouse brain slices. Intriguingly, these neurons displayed strikingly low levels and slow cAMP responsiveness compared to medium-sized spiny neurons (MSNs). Our experiments revealed that PDE1, PDE3 and PDE4 are important regulators of cAMP level in ChINs. Notably, the induction of cGMP production by nitric oxide (NO) donors increases cAMP by inhibiting PDE3 - a mechanism hitherto unexplored in neuronal context. Furthermore, the activation of NMDA or metabotropic glutamate receptors increases intracellular calcium, consequently activating PDE1 and thereby decreasing both cAMP and cGMP. This interplay of phosphodiesterases enables the control of cAMP by the neuromodulatory influences of glutamate and NO. Remarkably, the NO/cGMP signal results in different effects: NO enhances cAMP in ChINs by inhibiting PDE3, whereas it reduces cAMP levels in MSNs by activating PDE2A. These findings underscore the specificity of intracellular signaling in ChINs compared to MSNs and show how the NO-cGMP pathway affects these various neuronal types differently. These observations have significant implications for understanding the regulation of the striatal network and the integration of dopaminergic signals and suggest innovative therapeutic strategies for addressing basal ganglia disorders with unmet medical need.

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  1. Version published to 10.1101/2023.10.07.560741v2 on bioRxiv
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    Reply to the reviewers

    Reviewer #1 Evidence, reproducibility and clarity: Bompierre et al have presented a set of interesting findings that demonstrate the interconnected roles of multiple PDEs in striatal cholinergic interneurons. They show that the regulation of neurons by PDEs differs between ChiNs and MSNs. Disentangling the complex and interconnected biology of PDE subtypes and calcium signalling is notoriously difficult and I commend the authors for not shying away from it. The data are interesting and compelling however I have a number of readily addressable concerns regarding their statistical analysis.

    We are very grateful to the reviewer for the careful reading of our manuscript, positive comments and useful suggestions to enhance its readability. We also thank the reviewer for highlighting the difficulties resulting from the scarcity of these neurons. As described in the manuscript, during the course of several other projects, we noticed this sparse neuronal population that clearly departed from the vast majority of striatal neurons, the medium-sized spiny neurons. This particular neuronal population was identified as ChINs only later with immunohistochemistry. We were quite surprised that our visual identification was confirmed by immunohistochemistry in all cases, as described, now with more details, in the manuscript. We then performed a number of new experiments focused on ChINs while we could also re-analyze older experiments performed for other projects and in which ChINs were visually identified - all our experiments are terminated with the acquisition of a Z stack which allows a precise observation of each neuron in the imaging field. This explains some changes in the drugs presented in this manuscript.

    Major concerns: • Statistics, general comments: o When performing multiple comparisons (as in figures 2-6) the authors should be using a one-way ANOVA or Friedman's test (for non-parametric).

    Since data normality of a few samples was rejected by the Shapiro-Wilk test, and in line with our previous publication, we used non-parametric statistics for all of this study. The Friedman’s test is now applied to all situations comparing more than two conditions.

    o When comparing an effect size between experimental conditions e.g. cAMP following NMDA with or without Lu AF64193, the conditions need to be compared directly, not just inferred from being 0.05. [ see Makin and Orban de Xivry 2019 PMID: 31596231 and Nieuwnhuis et al 2011 PMID: 31596231 for more details].

    We agree with this comment and the recommended tests have been performed.

    o Please include P values and degrees of freedom for each analysis, rather than just * indicating PP values are now indicated in the text - although a smaller P value does not imply that the difference is “more” significant. Degrees of freedom do not apply with Friedman and Wilcoxon rank tests.

    • Introduction: o Page 3 para, describing the various PDEs reported to operate in ChiNs vs MSNs, is very convoluted and hard to follow. I recommend replacing this paragraph with a table: column 1: PDE subtype, 2: neuron type, 3: effect reported 4: ref.

    We agree that this paragraph was difficult to understand. We tried to build a table as suggested, but such table could not convey all the aspects that we think should be made clear (mRNA or protein data, for example). Instead, we propose a revised paragraph that we hope will be easier to read.

    • Methods: o Please include explanation for estimated cAMP concentration (right axis on figs 2c, 3b,5c-f

    This was described in “Methods / Estimates of biosensor activation level”. We also added a similar estimate for cGMP concentration based on our previous work with the cGMP biosensor cyGNAL in Figures 4 and 6.

    o Please specify slice thickness and age of mice

    Slice thickness was missing and now added in methods (300 µm). The age was indicated in the first paragraph of Methods: “7 to 12 days”.

    • Figure 1: o describe if 13 uM fsk is supposed to be a maximal concentration, what is the justification for this concentration. Is there a previously published dose-response curve?

    A biochemical study of adenylyl cyclase intracellular domains (VC1 and IIC2 heteromer) reports a Kd for forskolin of 0.1 µM {Dessauer et al., 1997, J Biol Chem, 272, 22272-7}, well below the dose we used. To our knowledge, there is no published dose-response analysis of forskolin effect in ChINs and we did not perform this measurement. We routinely use 13 µM for practical reasons, our stock being 25 mM diluted 2,000-fold. ChINs display a much lower cAMP response than MSNs at this dose: we think that this interesting observation deserves to be reported (but we would like to point out that this is only a marginal aspect of our study). We totally agree that this point deserves more explanation and a paragraph has been added in the discussion: “The low responsiveness of ChINs to cAMP-activating signals such as forskolin is striking but the underlying cellular mechanisms remain to be determined. All adenylyl cyclases except AC9 are activated by forskolin {Defer et al., 2000, Am J Physiol Renal Physiol, 279, F400-16}. AC1, AC2 and AC5 are widely expressed in the brain, including the striatum {Matsuoka et al., 1997, J Neurochem, 68, 498-506}. Cluster analysis of mRNA transcript suggest that AC1 and AC2 predominate in ChINs whereas AC5 is mainly expressed in MSNs {Saunders et al., 2018, Cell, 174, 1015-1030.e16}. This indicates that the reduced responsiveness to forskolin in ChINs compared to MSNs does not result from ChINs lacking adenylyl cyclases sensitive to forskolin.”.

    • Figure 2c: o Clarify which replicates are shown on the graph (I assume it's the n=11 i.e. number of slices)

    Yes, the plot shows the 11 replicates of the same protocol, with each set of 3 points of a same color linked with a line showing the measurements for one experiment. This is now stated more clearly in the figure legend.

    o You say "PDE3 thus contributes importantly to the regulation of cAMP level, and when this phosphodiesterase is inactivated pharmacologically, cAMP level becomes controlled by PDE4." But in fig 2c panel 2 inhibition of PDE4 with Picla increases cAMP without PDE3 being already inhibited. This doesn't agree with your statement, which implies that PDE4 only controls cAMP once PDE3 has been inhibited.

    This is unfortunate since we did not want to suggest that PDE4 has no effect unless PDE3 is inhibited. Indeed, this experiment together with the next actually demonstrate that PDE3 and 4 are simultaneously engaged in cAMP regulation. This ambiguity was also noted by reviewer 3. We thus rephrased the link between between these two paragraphs.

    o Data described but not shown: "In the absence of forskolin, application of PDE3 inhibitor (cilostamide, 1 µM, N=3; n=3; A=3) or PDE4 inhibitor (rolipram, 1 µM, N=2; n=2; A=2) or their combination (N=6; n=8; A=3) induced no significant ratio change in ChINs (as well as in MSNs)".

    We removed the mention to the experiments with cilostamide and rolipram alone since N was less than 5. The effect of rolipram and cilostamide together are displayed in Figure 2, with proper statistics indicated in the text.

    Also please explain why you have changed PDE inhibitor?

    This project developed over several years and the phosphodiesterase inhibitors used in the team changed depending on their availability. In addition, some data such as the lack of effects on baseline cAMP level were extracted from experiments performed for other purposes, hence some differences in the inhibitors we used. Please note that given the scarcity of this neuronal population, many more trials and mice would be required to reproduce these experiments with a single inhibitor. We further consider that our approach contributes to the desired reduction in the use of animals in research (the 3-R).

    • Figure 4: o Please clarify in text that data in 4b is from ChiNs and not pooled with data from MSNs? i.e. is the summary data from MSNs not shown?

    Figure 4B and C only show average calcium responses in ChINs. This is now indicated in the results section “Figure 4B shows the calcium response to NMDA in ChINs with the average trace (left), and baseline and peak amplitude (right) for individual ChINs.” The calcium response to NMDA uncaging in MSNs has already been published (Betolngar 2019) and is not shown in this study.

    o Response to DHPG is not formally compared between MSNs and ChiNs

    In MSNs, the calcium response to DHPG showed variability, with a lack of response in most experiments but a clear calcium signal in a few MSNs. The cause of this variability was not the focus of this study but, nevertheless, we wanted to mention this qualitative observation to stimulate future studies on this subject. However, if a quantitative measurement of this variability is required, the description of the DHPG effect on MSNs will be removed.

    • Figure 5: o Explain reasoning for switching PDE4 inhibitor (roflumilast).

    At the time these experiments were performed, we were using roflumilast to inhibit PDE4.

    Explain change in fsk concentration (0.5 uM in figure 5 vs 13 uM in earlier figs)

    We wanted to study PDE1 in isolation, i.e. in conditions in which both PDE3 and PDE4 were inhibited. Simultaneous inhibition of PDE3 and 4 leads to biosensor saturation (Figure 2), a situation in which a PDE1-mediated decrease in cAMP level might be difficult to resolve. The forskolin concentration was therefore reduced to decrease adenylyl cyclase activation. This is now explained in the manuscript: ”In order to stimulate a moderate cAMP production and thus maintain the visibility of PDE1 action, a lower concentration of forskolin (0.5 µM) was employed in these experiments.“

    o Text states: "These effects were blocked with Lu AF64196 (1-10 µM), a potent and selective PDE1 inhibitor" but this was not formally tested and in the figure a response is still visible (smaller than before Lu, but not blocked)

    Indeed, a small change in the average ratio trace is still visible, so we changed “blocked” by “largely reduced”. The effect of Lu AF64196 in the NMDA condition was tested as follows: the change in ratio level induced by NMDA uncaging was calculated in control and Lu AF64196 conditions. This ratio change was compared between control and Lu condition with a Wilcoxon rank test. The same test was applied to compare control and DHPG condition. This is now indicated in the manuscript.

    o Please explain the reason for the different time courses in figure 5ab vs 5c-f)

    Figure 5A,B are illustrative experiments. Figure 5C-F are average traces from several experiments. This is indicated in the figure legend.

    o Data not shown: "Of note, the addition of the PDE1 inhibitor did not increase the steady-state cAMP level, demonstrating that PDE1 did not exhibit a tonic activity before its activation by the calcium signal"

    In our experimental conditions, the cAMP level is too high to faithfully report an increase in cAMP upon Lu AF64196 application, so we removed this sentence. However, the cGMP level in the presence of DEANO is farther from saturation, allowing to perform this control: the application of Lu AF64196 produced no significant increase in cGMP level, indicating that PDE1 is not active in our experimental conditions. This has been added in the results.

    • Figure 6: o Comment on why using quisqualate (mixed AMPA and mGLuR) rather than DHPG as used previously?

    These early experiments were performed with these drugs until we made sure that group 1 mGlu was responsible for this effect and the more specific agonist DHPG was used. The drug combination, however, is specific of group I mGlu activation.

    o Again, effect reported as being blocked, but not formally tested and a response is still evident on the graph. If the authors believe that response is an artefact from the stim then please show data with NMDAR antagonists.

    We agree that a small change in the average ratio trace is still visible, so we changed “blocked” by “largely reduced”. The effect of Lu AF64196 was tested as described above for cAMP, which is now indicated in the manuscript. We agree that NMDA as well as mGlu stimulation, by increasing calcium, can affect cyclic nucleotides by other mechanisms than PDE1 activation. This was already reported in our previous work, in particular in the hippocampus and cortex (Betolngar 2019). We are not sure that NMDAR antagonists would clarify the situation since NMDA receptor blockade would probably suppress the cAMP change that is still visible in the presence of the PDE1 inhibitor. Nonetheless our manuscript reports experimental conditions in which the vast majority of the effect that we focus on is blocked by the PDE1 selective inhibitor.

    • Discussion: o Add references in para 2 describing the PDEs expressed by ChiNs

    Done

    o Figure 7: cartoon indicates that calcium will exclusively activate PDE1A, is this for simplicity or is their evidence to support this?

    Single-cell RT-PCR data {Saunders et al., 2018, #42891} indicate that ChINs express PDE1A but not PDE1B. This is now indicated in the introduction. The cartoon has been changed accordingly.

    o Please comment on the specificity of the pharmacological tools used throughout the study.

    Phosphodiesterases bear a cleft-shaped catalytic site that is particularly amenable to chemical inhibition, and phosphodiesterase thus constitute a therapeutic target of great interest: a large number of highly specific phosphodiesterase inhibitors have been developed and tested by pharmaceutical companies. It nonetheless remains that our demonstration relies on the specificity of the phosphodiesterase inhibitors. We added a sentence in the discussion “We used highly specific phosphodiesterase inhibitors to acutely test the functional contribution of these phosphodiesterases.” to acknowledge this.

    o In the context of regulation of striatal cholinergic and dopaminergic signalling by NO (via cGMP), I believe the authors should cite Hartung et al PMID: 21508928

    This very valuable citation has been added in the discussion.

    Minor comments: • Page 3 paragraph 1 and 3 the authors have used ellipsis instead of finishing their sentences.

    Done.

    • In figure 1a please indicate you are recording in dorsomedial region of the striatum (you state in methods this is your recording location, but it would be helpful to show it here)

    We thought of improving the cartoon in Figure 1 by drawing a rectangle over the dorso-medial striatum on the image of the brain slice, but that would suggest that the slices had been cut at this stage of the preparation, which was not the case. Adding another image of a brain slice would clutter the figure. We leave it to the Editor to decide how this should be handled.

    • The authors use a lot of different drugs, I think a table listing the drugs, concentrations and their targets would help limit confusion.

    This will certainly make it easier for the reader. This table has been added in Methods / Chemicals and drugs.

    • I found the graphs with each replicate in a different colour quite difficult to read. My personal preference would be for graphs to show each replicate as a transparent line/small symbol, with the mean and SEM shown larger and in bold.

    We tried to enhance the visibility as suggested. SEM should not be shown since our statistics are non-parametric.

    Significance: General strengths: The question of how PDEs interact to regulate striatal output is extremely interesting and notoriously difficult to tackle. The authors have relied upon pharmacological manipulation of PDEs. A pharmacological approach has both strengths (intact system with little compensation occurring between PDEs, which would occur with a genetic strategy) and weaknesses (relying on each of the drugs to act selectively and specifically). By investigating multiple PDEs in the same system in two neuron types, I believe the authors are illustrating interesting findings. For these findings to be more concrete I believe they need a more robust statistical approach, but the experiments and questions are valid. PDEs are of interest to most neuroscientists and understanding cholinergic function is of interest to anyone studying the striatum of basal ganglia more broadly. My expertise is investigating the interactions between striatal cholinergic and dopaminergic signalling.

    Reviewer #2 Evidence, reproducibility and clarity : The work characterizes in depth the dynamics of the cyclic nucleotide signaling in cholinergic interneurons (ChINs) in the striatum and the interconnection with calcium signaling. The study is ambitious and risky since it targets a minority of neurons representing only 1% of the total population of striatal neurons. For that they used genetically encoded biosensors, at a very low infection rate, and highly specific phosphodiesterase inhibitors. With these tools they defined PDE1, PDE3 and PDE4 as the key regulators of cAMP levels in ChINs and the interplay with incoming signals raising cGMP and free-calcium levels after nitric oxide or glutamate activation of NMDA or mGlu1/5 receptors, respectively. The conclusions of the study are solid and well supported by the experimental results.

    We would like to thank the reviewer for his/her positive comments about our work.

    The team has the necessary technical and conceptual background in the field. This is very important to trust the criteria they used to identify ChINs, a fundamental hallmark in this study.

    Again, we are very grateful to the reviewer for this very positive comment.

    Still, confirmation by immunohistochemical labeling with ChAT antibodies sounds important and perhaps it should had been performed in more experiments.

    We agree that our qualitative immunostaining validation of ChAT expression was too terse. We re-analyzed our archived data to provide a more precise account of our observations. We first identified ChINs from their morphology in the biosensor image stack, then checked whether these neurons were positive for ChAT. This is now explained in detail in “Identification of Cholinergic Interneurons in a brain slice”: “11 brain slices were fixed after the biosensor experiment and later processed for ChAT immunoreactivity. In these slices, 15 neurons were visually identified as ChINs during the biosensor recording session. All of these neurons showed a positive ChAT labelling.”

    Significance: The results represent an important conceptual advance in the field. To understand better the signaling that regulates firing of cholinergic neurons in the striatum might be relevant to explain pathological responses and they could be useful to define better strategies for the treatment of Parkinson's patients, for instance. In this regard, this study fills an existing gap since this elusive neuronal population was not functionally characterized before. The basic aspects of the study could be of interest to a broad audience.

    Reviewer #3 Evidence, reproducibility and clarity: Summary: The author's present very elegant findings regarding how NO regulates cAMP in striatal cholinergic interneurons (ChINs). The major strength of the manuscript lies in the approach, which enables single-cell imaging of neuronal signaling in acute brain slices. A clever combination of pharmacological tools were then utilized to dissect PDE contribution toward cAMP alterations in ChINs. While the manuscript is high-quality, there are a few controls and points of discussion that need to be considered.

    We would like to thank the reviewer for his/her careful reading of our manuscript and very positive comments about our study.

    Major: There are numerous references to results seemingly missing from the figures.

    • Figure 2 TP-10
    • Figure 2 PF-05 traces
    • Figure 2 data in absence of FSK (rolipram, cilo)
    • Figure 3 ODQ

    We thought that these experiments showing a lack of effect were of little interest to the reader and therefore omitted raw traces and statistics from the manuscript. However, we fully agree to display more of our data, as long as it does not clutter the main points of our manuscript. We now illustrate the lack of effect of PDE2A inhibition with a typical experiment in Figure 2C. The ratio level is now shown in Figure 2D for roli-cilo and TP-10, with matching statistics in the text. Figure 3 now shows the lack of effect of ODQ.

    Have the author's considered an alternative perspective that slow cAMP detection in ChIN, relative to MSN, could be due to the size of neuron? The significantly greater volume of ChIN soma could conceivably require more cAMP to reach the detection threshold of the biosensor. Therefore, how do the author's reconcile such technical caveats?

    This is a very interesting hypothesis that is supported by many theoretical and experimental data: it takes longer for membrane adenylyl cyclases to fill up a void volume in which both phosphodiesterases and the biosensor reside. We could rule-out the buffering effect of the biosensor by the experiment described in Figure 1B, but a lower surface to volume ratio such as that observed for ChINs vs MSNs could indeed explain a biologically slower onset in cAMP level. However, a lower surface to volume ratio should not affect the steady-state level that will be eventually reached upon continuous forskolin application: it takes longer to fill up the volume but, if waiting long enough, the final level will be only determined by the equilibrium between cyclase and phosphodiesterase. Forskolin applications of more than 10 min (Figure 2) led to steady-state levels that were far below biosensor saturation, while it did reach saturation in MSNs. Therefore, while we certainly acknowledge the importance of the peculiar neuronal morphology of ChINs, there must be additional specific differences between ChINs and MSNs. In any case, we agree that this important point was missing in our manuscript and we added a paragraph in the discussion to discuss differences in adenylyl cyclases and cell geometry.

    In the traces from Figure 2, it is unclear why PDE3 and PDE4 have differential contributions toward cAMP elevation depending on the order of inhibition.

    Thank you for pointing out our poor wording, which has also been noted by the first reviewer. Indeed, the data in Figure 2 shows that PDE3 and PDE4 are simultaneously engaged in cAMP regulation. This part has been rewritten.

    Moreover, we cannot conclude that PDE3 and PDE4 the major PDEs in ChIN from such experiment based on the result that IBMX did not further raise cAMP. Likely, the biosensor has reached the detection ceiling. This should be discussed as a possibility.

    It is an interesting possibility that cAMP levels higher than biosensor saturation level ([cAMP] above 100 µM) could be modulated after PDE3 and PDE4 inhibition, in concentration ranges that go beyond biosensor saturation level. However, our experiments clearly demonstrate that the combined action of PDE3 and PDE4 constitutes the first line of cAMP control since the concomitant inhibition of PDE3 and PDE4 raises [cAMP] beyond physiological relevance. When both PDE3 and PDE4 were inhibited, cAMP indeed reached the level of biosensor saturation which led us to state “The ratio was not further raised by the non-specific phosphodiesterase inhibitor IBMX (200 µM), indicating the saturation level of the biosensor by cAMP (Rmax)”, a conclusion that remains valid.

    The author's intepretation ignores the influence of calcium on the activity of various types of ACs, which seems to be a critical feature given the experimental design that first broadly stimulates ACs with forskolin.

    We agree with the Reviewer that calcium will certainly activate AC1 (possibly present in ChINs) and inhibit AC5 (expressed in MSNs and possibly also in ChINs). Our experimental design relies on the highly specific PDE1 inhibitor to isolate the selective contribution of PDE1, but minor changes in cAMP and cGMP remained even after PDE1 inhibition, as also pointed out by the first Reviewer. We found experimental conditions in which the contribution of PDE1 could be largely visible, which was the point of this study. However, we certainly agree with the Reviewer that a calcium signal will affect cyclic nucleotide levels through a number of other mechanisms. Therefore, we added the sentence “It should also be noted that, in the presence of the PDE1 inhibitor Lu AF64196, some changes in cAMP level still remained, which can result either from incomplete PDE1A inhibition and/or from NMDA effects on other targets, such as calcium-modulated adenylyl cyclases.”

    A missing control in Figure 5 is the effect of Lu AF64196 by itself on cAMP (and in the presence of FSK pre stimulation).

    We agree that this is an important control. However, this could not be tested on this protocol with cAMP since the ratio was too close to Rmax, and an increase in cAMP level following PDE1 inhibition would be undetectable. However, with cGMP imaging, the ratio reached with DEANO was farther from saturation such that an increase in cGMP resulting from the inhibition of PDE1 should be detectable. However, Lu AF64196 showed no significant effect. This measurement was added in the manuscript: “As a control, we verified that PDE1inhibition had no effect on the steady-state cGMP level elicited by 10 µM DEANO (Figure 7G: in DEANO: 0.78 of Rmax; in DEANO and Lu AF64196: 0.81; N=5, n=6, a=5; Wilcoxon P=0.094).”

    The mechanism would be signicantly bolstered by measuring cAMP from PDE4 inhibition following forskolin and DEANO (Figure 3).

    It is true that, in the presence of forskolin and DEANO, the only PDE that remains is PDE4: its inhibition should increase cAMP to the maximal level. Unfortunately, this experimental scheme was not tested. However, Figure 3 is focusing on the regulation of PDE3 by cGMP and at this stage of the reasoning, it might be confusing to get back to the question of which PDE remains after PDE3 has been blocked. In this manuscript, we want to highlight that PDE3 is blocked via the NO-cGMP signaling pathway, and we think that the data in Figure 3 demonstrates this point clearly.

    Minor: The introduction should discuss the critical role of ChIN in striatum rather than simply stating "critical role in striatal functions"

    A paragraph has been added in the Introduction to highlight the importance of ChINs in striatal function.

    PDE should be included as abbreviation in introduction after first mention of phosphodiesterases.

    We agree that this was inducing confusion. We keep PDE1-11 as it is the official names of proteins, but we replaced all occurrences of “PDE” by “phosphodiesterases” when alluding to the general concept of this class of enzymes.

    Light sources (e.g. laser) for excitation during imaging are missing from the methods.

    This was indicated in Methods / Biosensor imaging: “LED light sources (420 nm with a 436 nm excitation filter and 360 nm) were purchased from Mightex (Toronto, Canada).”

    The study certainly provides implication for diseases associated with striatal dysfunction such as Parkinson's disease. However, it may be important to note that experiments were performed on slice preparations from very young animals, which could have inherent differences in functionality relative to an aged or diseased context.

    We agree that our preparation of brain slices from mice pups is not representative of what could be found in adults, and even less in pathological conditions. Nonetheless, we believe that we identified a crosstalk mechanism that has never been reported in neurons, and that is not taken into account in theories of striatal functions. We hope that this novel understanding will lead to the development of experiments on adult mice that might confirm the functional importance of this effect, in particular pharmacological studies in adult animals with PDE3 inhibitors.

    Significance: General assessment: The study utilizes pharmacological tools to selectively target enzymes and receptors in the cAMP cascade to mechanistically dissect how cAMP is handled in ChINs. The major strength is ability to perform such experiments in acute brain slices, i.e. a "native" neuronal context. An exciting aspect is stimulation of NMDAR by agonist-uncaging, thereby revealing an endogenous signaling route that modulates cAMP. A major physiological limitation is reliance on fsk to induce AC activity, however the approach is suitable to obtain mechanistic information. Moreover, conducting experiments in a Parkinsonian disease model would provide tremendous value, although such pursuits are beyond the scope of the work here. Advance: The study builds off robust studies previously published by the author's. The work is also similar to AC-cAMP investigations on acute brain slices performed by the Sabatini (24765076, 29154125) and Martemyanov (29298426, 31644915) labs, which unfortunately were not discussed/cited here. This is perhaps a missed opportunity to highlight the significance of the author's study. For instance, the Sabatini lab investigated calcium influence on cAMP but in the hippocampus. The Martemyanov lab investigated striatal cAMP, but not in ChINs or through calcium or cGMP mechanisms. Therefore there is a gap toward understanding striatal ChINs, which is clearly demonstrated in the author's work here.

    These very important references have been left out of our manuscript intentionally since not pertaining directly to the topic of the manuscript. We would like to point out that we also omitted citations to our own work which had also described dopamine, adenosine and acetylcholine responses measured with various biosensors in striatal neurons (Castro 2013 23551948; Yapo 2017 28782235; Nair 2019 24560149). Indeed, this research field is flourishing and we hope that people interested in the general question of cAMP signal integration in neurons will easily find many such relevant publications.

    Audience: I find this basic research to have a relatively broad appeal. For example, my lab is working on behavioral aspects of motor dysfunction. Therefore, the pharmacological insight here is very intriguingly. The mechanistic nature of the work may also appeal to those working on the signaling aspect of such diseases.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    The author's present very elegant findings regarding how NO regulates cAMP in striatal cholinergic interneurons (ChINs). The major strength of the manuscript lies in the approach, which enables single-cell imaging of neuronal signaling in acute brain slices. A clever combination of pharmacological tools were then utilized to dissect PDE contribution toward cAMP alterations in ChINs. While the manuscript is high-quality, there are a few controls and points of discussion that need to be considered.

    Major:

    There are numerous references to results seemingly missing from the figures.

    • Figure 2 TP-10
    • Figure 2 PF-05 traces
    • Figure 2 data in absence of FSK (rolipram, cilo)
    • Figure 3 ODQ

    Have the author's considered an alternative perspective that slow cAMP detection in ChIN, relative to MSN, could be due to the size of neuron? The significantly greater volume of ChIN soma could conceivably require more cAMP to reach the detection threshold of the biosensor. Therefore, how do the author's reconcile such technical caveats?

    In the traces from Figure 2, it is unclear why PDE3 and PDE4 have differential contributions toward cAMP elevation depending on the order of inhibition. Moreover, we cannot conclude that PDE3 and PDE4 the major PDEs in ChIN from such experiment based on the result that IBMX did not further raise cAMP. Likely, the biosensor has reached the detection ceiling. This should be discussed as a possibility.

    The author's intepretation ignores the influence of calcium on the activity of various types of ACs, which seems to be a critical feature given the experimental design that first broadly stimulates ACs with forskolin.

    A missing control in Figure 5 is the effect of Lu AF64196 by itself on cAMP (and in the presence of FSK pre stimulation).

    The mechanism would be signicantly bolstered by measuring cAMP from PDE4 inhibition following forskolin and DEANO (Figure 3).

    Minor:

    The introduction should discuss the critical role of ChIN in striatum rather than simply stating "critical role in striatal functions"

    PDE should be included as abbreviation in introduction after first mention of phosphodiesterases.

    Light sources (e.g. laser) for excitation during imaging are missing from the methods.

    The study certainly provides implication for diseases associated with striatal dysfunction such as Parkinson's disease. However, it may be important to note that experiments were performed on slice preparations from very young animals, which could have inherent differences in functionality relative to an aged or diseased context.

    Significance

    General assessment:

    The study utilizes pharmacological tools to selectively target enzymes and receptors in the cAMP cascade to mechanistically dissect how cAMP is handled in ChINs. The major strength is ability to perform such experiments in acute brain slices, i.e. a "native" neuronal context. An exciting aspect is stimulation of NMDAR by agonist-uncaging, thereby revealing an endogenous signaling route that modulates cAMP. A major physiological limitation is reliance on fsk to induce AC activity, however the approach is suitable to obtain mechanistic information. Moreover, conducting experiments in a Parkinsonian disease model would provide tremendous value, although such pursuits are beyond the scope of the work here.

    Advance:

    The study builds off robust studies previously published by the author's. The work is also similar to AC-cAMP investigations on acute brain slices performed by the Sabatini (24765076, 29154125) and Martemyanov (29298426, 31644915) labs, which unfortunately were not discussed/cited here. This is perhaps a missed opportunity to highlight the significance of the author's study. For instance, the Sabatini lab investigated calcium influence on cAMP but in the hippocampus. The Martemyanov lab investigated striatal cAMP, but not in ChINs or through calcium or cGMP mechanisms. Therefore there is a gap toward understanding striatal ChINs, which is clearly demonstrated in the author's work here.

    Audience:

    I find this basic research to have a relatively broad appeal. For example, my lab is working on behavioral aspects of motor dysfunction. Therefore, the pharmacological insight here is very intriguingly. The mechanistic nature of the work may also appeal to those working on the signaling aspect of such diseases. Please define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

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    Referee #2

    Evidence, reproducibility and clarity

    The work characterizes in depth the dynamics of the cyclic nucleotide signaling in cholinergic interneurons (ChINs) in the striatum and the interconnection with calcium signaling. The study is ambitious and risky since it targets a minority of neurons representing only 1% of the total population of striatal neurons. For that they used genetically encoded biosensors, at a very low infection rate, and highly specific phosphodiesterase inhibitors. With these tools they defined PDE1, PDE3 and PDE4 as the key regulators of cAMP levels in ChINs and the interplay with incoming signals raising cGMP and free-calcium levels after nitric oxide or glutamate activation of NMDA or mGlu1/5 receptors, respectively. The conclusions of the study are solid and well supported by the experimental results. The team has the necessary technical and conceptual background in the field. This is very important to trust the criteria they used to identify ChINs, a fundamental hallmark in this study. Still, confirmation by immunohistochemical labeling with ChAT antibodies sounds important and perhaps it should had been performed in more experiments.

    Significance

    The results represent an important conceptual advance in the field. To understand better the signaling that regulates firing of cholinergic neurons in the striatum might be relevant to explain pathological responses and they could be useful to define better strategies for the treatment of Parkinson's patients, for instance. In this regard, this study fills an existing gap since this elusive neuronal population was not functionally characterized before. The basic aspects of the study could be of interest to a broad audience.

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    Referee #1

    Evidence, reproducibility and clarity

    Bompierre et al have presented a set of interesting findings that demonstrate the interconnected roles of multiple PDEs in striatal cholinergic interneurons. They show that the regulation of neurons by PDEs differs between ChiNs and MSNs. Disentangling the complex and interconnected biology of PDE subtypes and calcium signalling is notoriously difficult and I commend the authors for not shying away from it. The data are interesting and compelling however I have a number of readily addressable concerns regarding their statistical analysis.

    Major concerns:

    • Statistics, general comments:
      • When performing multiple comparisons (as in figures 2-6) the authors should be using a one-way ANOVA or Friedman's test (for non-parametric).
      • When comparing an effect size between experimental conditions e.g. cAMP following NMDA with or without Lu AF64193, the conditions need to be compared directly, not just inferred from being < or > 0.05. [ see Makin and Orban de Xivry 2019 PMID: 31596231 and Nieuwnhuis et al 2011 PMID: 31596231 for more details].
      • Please include P values and degrees of freedom for each analysis, rather than just * indicating P<0.05
    • Introduction:
      • Page 3 para, describing the various PDEs reported to operate in ChiNs vs MSNs, is very convoluted and hard to follow. I recommend replacing this paragraph with a table: column 1: PDE subtype, 2: neuron type, 3: effect reported 4: ref.
    • Methods:
      • Please include explanation for estimated cAMP concentration (right axis on figs 2c, 3b,5c-f
      • Please specify slice thickness and age of mice
    • Figure 1:
      • describe if 13 uM fsk is supposed to be a maximal concentration, what is the justification for this concentration. Is there a previously published dose-response curve?
    • Figure 2c:
      • Clarify which replicates are shown on the graph (I assume it's the n=11 i.e. number of slices)
      • You say "PDE3 thus contributes importantly to the regulation of cAMP level, and when this phosphodiesterase is inactivated pharmacologically, cAMP level becomes controlled by PDE4." But in fig 2c panel 2 inhibition of PDE4 with Picla increases cAMP without PDE3 being already inhibited. This doesn't agree with your statement, which implies that PDE4 only controls cAMP once PDE3 has been inhibited.
      • Data described but not shown: "In the absence of forskolin, application of PDE3 inhibitor (cilostamide, 1 µM, N=3; n=3; A=3) or PDE4 inhibitor (rolipram, 1 µM, N=2; n=2; A=2) or their combination (N=6; n=8; A=3) induced no significant ratio change in ChINs (as well as in MSNs)". Also please explain why you have changed PDE inhibitor?
    • Figure 4:
      • Please clarify in text that data in 4b is from ChiNs and not pooled with data from MSNs? i.e. is the summary data from MSNs not shown?
      • Response to DHPG is not formally compared between MSNs and ChiNs
    • Figure 5:
      • Explain reasoning for switching PDE4 inhibitor (roflumilast). Explain change in fsk concentration (0.5 uM in figure 5 vs 13 uM in earlier figs)
      • Text states: "These effects were blocked with Lu AF64196 (1-10 µM), a potent and selective PDE1 inhibitor" but this was not formally tested and in the figure a response is still visible (smaller than before Lu, but not blocked)
      • Please explain the reason for the different time courses in figure 5ab vs 5c-f)
      • Data not shown: "Of note, the addition of the PDE1 inhibitor did not increase the steady-state cAMP level, demonstrating that PDE1 did not exhibit a tonic activity before its activation by the calcium signal"
    • Figure 6:
      • Comment on why using quisqualate (mixed AMPA and mGLuR) rather than DHPG as used previously?
      • Again, effect reported as being blocked, but not formally tested and a response is still evident on the graph. If the authors believe that response is an artefact from the stim then please show data with NMDAR antagonists.
    • Discussion:
      • Add references in para 2 describing the PDEs expressed by ChiNs
      • Figure 7: cartoon indicates that calcium will exclusively activate PDE1A, is this for simplicity or is their evidence to support this?
      • Please comment on the specificity of the pharmacological tools used throughout the study.
      • In the context of regulation of striatal cholinergic and dopaminergic signalling by NO (via cGMP), I believe the authors should cite Hartung et al PMID: 21508928

    Minor comments:

    • Page 3 paragraph 1 and 3 the authors have used ellipsis instead of finishing their sentences.
    • In figure 1a please indicate you are recording in dorsomedial region of the striatum (you state in methods this is your recording location, but it would be helpful to show it here)
    • The authors use a lot of different drugs, I think a table listing the drugs, concentrations and their targets would help limit confusion.
    • I found the graphs with each replicate in a different colour quite difficult to read. My personal preference would be for graphs to show each replicate as a transparent line/small symbol, with the mean and SEM shown larger and in bold.

    Significance

    General strengths: The question of how PDEs interact to regulate striatal output is extremely interesting and notoriously difficult to tackle. The authors have relied upon pharmacological manipulation of PDEs. A pharmacological approach has both strengths (intact system with little compensation occurring between PDEs, which would occur with a genetic strategy) and weaknesses (relying on each of the drugs to act selectively and specifically).

    By investigating multiple PDEs in the same system in two neuron types, I believe the authors are illustrating interesting findings. For these findings to be more concrete I believe they need a more robust statistical approach, but the experiments and questions are valid. PDEs are of interest to most neuroscientists and understanding cholinergic function is of interest to anyone studying the striatum of basal ganglia more broadly. My expertise is investigating the interactions between striatal cholinergic and dopaminergic signalling.

  6. Version published to 10.1101/2023.10.07.560741v1 on bioRxiv