PONE-D-19-30235

Converging sensory and motor cortical inputs onto the same striatal neurons: an in vivo intracellular investigation

PLOS ONE

Dear Pr. Charpier,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The paper by Charpier et al describes intracellular recordings from neurons in the dorsolateral striatum of anesthetized rats. The authors show that striatal projection neurons (MSNs) responding to contralateral whisker deflections also respond to electrical stimulation of the ipsilateral motor cortex, thus suggesting that they may be involved in sensorimotor processes. The authors also confirm that these responses to electrical stimulation are monosynaptic, based on the onset latencies in striatal and cortical neurons. The manuscript is interesting, timely, and well-written, and the data presented are convincing. I have just a few comments regarding additional information to be added and the relationship to previously published studies.

Sensorimotor targeting: The targeting of MSNs by sensory and motor cortices, even in overlapping regions, was shown in earlier papers using other methods: (Wall…Kreitzer, Neuron (2013), Reig & Silberberg, Cerebral cortex (2016), ). Moreover, it was shown that both types of MSNs (direct and indirect pathway neurons) receive these inputs. The issue of selective targeting of direct and indirect pathways should be addressed, especially as the data in the paper showing that a vast majority of recorded MSNs respond to both types of cortical inputs. These data strongly suggest that both MSN types respond to both types of cortical stimulation.

Was there any attempt to explain the diversity in response by staining for D1/D2/SP/ENK to determine dMSN or iMSN identity of recorded neurons?

Another comparison would be to assess the difference between S1 and M1 responses under the same conditions. Currently, the stimulation of S1 is done indirectly by whisker deflection while M1 is activated electrically. Do the authors have comparable data showing MSN responses to electrical stimulation in S1 compared to M1? If such electrical stimuli are given with same parameters, one could conclude whether MSNs in dorsolateral striatum receive stronger or weaker inputs from these regions.

Bilateral projections: previous studies (Brown et al., Neuroscience (1996); Lei et al, J.Neuroscience (2004); Reig and Silberberg, cerebral cortex (2016)) have shown that one of the main differences between corticostriatal projections from primary sensory and motor cortices is the density of contralateral innervation. Projections to contralateral striatum from primary sensory cortex are much sparser than those from motor cortex and other more frontal regions. Do the authors have any data regarding bilateral whisker and/or motor cortex stimulation? One interesting question that could be answered using the experimental setup is the comparison between ipsilateral and contralateral electrical and sensory stimulation. The prediction, based on previous data is that such differences will be more pronounced in the sensory responses than motor stimulation.

Feedforward inhibition: the role of inhibitory connections within the striatum (mainly striatal interneurons but also MSN collaterals) was shown to be instrumental in shaping both spontaneous activity and whisker evoked responses (Reig & Silberberg, neuron 2014). Moreover, inputs to striatal interneurons (in particular FS interneurons) were shown to arise from various cortical regions, including sensory, motor and others. This suggests that the responses described in this study (both sensory and electrical stimuli) are shaped by an inhibitory component as well. This issue should be addressed, at least in discussing the cortical inputs, and if possible also in presentation of experimental data. Such an inhibitory component could be revealed by depolarizing the MSNs recorded intracellularly.

Minor:

The authors show that 87% of recorded neurons respond to whisker stimulation. This is significantly higher than their earlier paper (2011) that showed 55% of responding neurons. What could be the reason for this difference in responsiveness? Was there any difference in the recording region or stimulus presentation?

The order of authors is different in the paper heading and in the cover pages (2nd and 3rd authors).

Results page 8: “…relatively weak Rm…”. Better to use high/low Rm.

Reviewer #2: In this manuscript, Charpier et al., investigated if projection neurons in the dorsolateral region of the striatum respond to both cortical input from functionally-related sensory and cortical regions. A key aspect of this study is that it is conducted in vivo, using intracellular electrophysiological recordings of striatal projection neurons (76 in total, which is a large number, considering the technical challenge of such approach), thus allowing to monitor supra et subthreshold changes in the membrane potential, in response to cortical/whisker stimulation. First, the authors reported that a large proportion of the striatal neurons recorded respond to whisker stimulation either through sub-threshold or supra-threshold depolarizing potentials. EcoG and intracell recordings in the barrel cortex provide strong evidence that whisker-evoked responses in the striatum were cortically driven. Then the authors showed that the same neurons also responded to electrical stimulation of M1 and provided evidence that these responses were mono-synaptically driven (rather than secondary to intra-cortical stimulation of S1). A key experiment was to record simultaneously S1 and striatal neurons while stimulating M1. The authors reported that striatal neurons responded with a shorter latency than S1 to M1 stimulation.

Altogether the authors provide excellent and rare in vivo electrophysiolgical data that demonstrate that the same striatal projection neurons can respond to sensory and motor cortical input from a functionally-related brain region. This knowledge is critical to understand the function of this brain region.

The study was carefully and rigorously conducted and the results which are in agreement with previous anatomical studies, obtained in vivo, constitute an important piece of information that should be shared with the community.

I have a few minor suggestions that the author may consider to address to improve their manuscript.

1) I was surprised by the opening sentence of the manuscript and the references cited. The authors mentioned the role of the striatum production and optimization of context-dependant behavior (isn't this the purpose of the whole nervous system?) and cite relatively “old” reviews of Ann Graybiel, which postulate a role of the striatum in motor control akin to as sort of traffic light signaling the beginning and end of automatized motor sequences, and thus overlook the moment to moment sensory representation associated with any behavior.

In my opinion, there are studies from the Silberberg and West labs that could have been provided a better context for this study to emphasis the importance of sensorimotor integration in the striatum (see also Robbe 2018 for a recent review on this topic)

2) A recent detailed anatomical tracing study nicely illustrate that vibrissal M1 and S1 send convergent input into specific region of the striatum (Hooks et al., 2018) and that this convergence does not occur for functionnaly distinct cortical area. This work should be cited

3) In the introduction, the authors suggested that sensory information is refined in the striatum. But from the description provided one could very well conclude that the information is degraded in the striatum. In fact both interpretation (refinement vs degradation or even filtering) could be correct. Maybe the author should use a more neutral word than “refinement” ?

4) When reading the result section for the first time it was not clear to me if the neurons recorded while stimulating M1 where the same one than those for which whisker responsiveness had been tested. The authors should make it very clear in the text (p10, after subsection title) that these are the same neurons.

5) The authors recorded 76 projection neurons. Most likely half of them where striatopallidal neurons while the other half. There has been anatomical evidence that striatonigral (direct-pathway) neurons are more strongly connected with the sensory cortex while striatopallidal (indirect-pathway) neurons are more connected with the motor cortex. It would have been nice if the authors would have speculated on the possible dichotomy of responses (or absence of) between these two populations.

6) I am not sure the authors should use a t-test to compare latencies between responses of striatal and S1 neurons (figure 5). The number of S1 is small (n=8) and I doubt normality can be evaluate. A non parametric test would be more appropriate, even if I have no doubt the result will be the same.

David Robbe

Reviewer #3: In this manuscript, the authors examine and characterize the cortico-striatal inputs from M1 and S1 to MSNs of the dorsolateral striatum (DLS) during whisker-induced sensory processing. Their major aim is to evaluate whether M1 and S1 inputs may converge on the very same MSN in this striatal area. The authors used an approach combining S1 and MSN intracellular recording and S1 electrocorticogram in vivo in anaesthetized rats subjected to whisker stimulation. The data showed an heterogenous response of DLS-somatosensory MSN to whisker sensory stimuli, and indirectly demonstrated a short-lasting direct monosynaptic M1 to MSN DLS connection. The data also demonstrated that sensory evoked whisker stimuli elicit responses in MSNs which are also responsive to M1 input, confirming their hypothesis of a M1 - S1 input convergence on individual MSNs in the DLS.

The question addressed in this paper is of high interest in the field to more deeply understand the motor and somatosensory input processing in the striatum. The experimental design providing in vivo recordings in DLS and the S1 cortex in a paradigm of sensory stimulation is highly adequate and perfectly conducted, and, the manuscript is clearly written with figures and text describing the results accurately. It delivers data that reinforce the knowledge on striatal computation of diverse cortical inputs. The following minor concerns must be considered.

The authors acknowledged the fact that CS inputs activation in their paradigms may or should result in both direct excitatory activation and feedforward inhibition of MSN, through activation of interneurons (see the discussion on the increase in synaptic conductance). However, no experiments were performed to evaluate the contribution of this feedforward inhibition in the protocols used in this study. Intrastriatal pharmacological GABAergic blockade could help to identify this contribution and this could be therefore clarified.

The data examining the membrane conductance of MSNs during the M1-induced response as illustrated in fig 4D should be more clearly presented. Indeed, the negative current pulse has a duration of 5ms in case of conjunction with the M1-induced responses (bottom traces) whilst it is of hundreds of ms in absence of stimulation (top trace). Moreover, calculation and comparison of conductances in both conditions should be provided.

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Reviewer #1: No

Reviewer #2: Yes: David Robbe

Reviewer #3: No

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Converging sensory and motor cortical inputs onto the same striatal neurons: an in vivo intracellular investigation

PONE-D-19-30235R1

Dear Dr. Charpier,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Jean-Pierre Mothet, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have addressed my comments in the discussion section of the paper. No additional data has been added but I hope will be done in future studies.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: Yes: David Robbe

Reviewer #3: No