Characterising the influence of cerebellum on the neuroplastic modulation of intracortical motor circuits.

The cerebellum (CB) has extensive connections with both cortical and subcortical areas of the brain, and is known to strongly influence function in areas it projects to. In particular, research using non-invasive brain stimulation (NIBS) has shown that CB projections to primary motor cortex (M1) are likely important for facilitating the learning of new motor skills, and that this process may involve modulation of late indirect (I) wave inputs in M1. However, the nature of this relationship remains unclear, particularly in regards to how CB influences the contribution of the I-wave circuits to neuroplastic changes in M1. Within the proposed research, we will therefore investigate how CB effects neuroplasticity of the I-wave generating circuits. This will be achieved by downregulating CB excitability while concurrently applying a neuroplastic intervention that specifically targets the I-wave circuitry. The outcomes of this study will provide valuable neurophysiological insight into key aspects of the motor network, and may inform the development of optimized interventions for modifying motor learning in a targeted way.

Introduction

The ability to modify patterns of motor behaviour in response to sensory feedback represents a fundamental component of effective motor control. This process underpins our capacity to learn new types of motor skills, and to improve their performance with practice. While this error-based motor adaptation is a complex process involving a distributed brain network, extensive literature has shown that the cerebellum (CB) plays a critical role (for review, see; [1]). This structure is thought to facilitate generation and ongoing modification of internal models of neural activation that determine effector dynamics. These internal models are constantly updated based on comparisons between predicted and actual sensory feedback, allowing improved task performance with practice. As an extension of this process, communication between CB and primary motor cortex (M1) is crucial [2, 3], and may facilitate retention of the generated internal model [4]. However, the neurophysiological processes underpinning this communication remain unclear, largely due to the difficulty of assessing the associated pathways in human participants.

Despite this, non-invasive brain stimulation techniques (NIBS) such as transcranial magnetic stimulation (TMS) have provided some information on CB-M1 communication. In particular, inhibitory interactions between CB and M1 have been demonstrated using a paradigm called CB-brain inhibition (CBI). This involves applying a TMS pulse over the CB at specific intervals (5–7 ms) prior to a second stimulus over contralateral M1, producing a motor evoked potential (MEP) that is reduced in amplitude relative to an MEP produced by M1 stimulation alone [5-7]. CBI is thought to involve activation of purkinje cells in CB cortex, leading to inhibition of the dentate nucleus and consequent disfacilitation of M1 via projections through the motor thalamus (for review, see; [8]). Activity of this pathway is known to be modified during the learning of adaptation tasks that rely heavily on input from the CB [9-11], with larger changes in CBI predicting better performance [11]. Furthermore, manipulating activity in the CB-M1 pathway can influence neuroplasticity in M1 [12-14]. Consequently, CB-M1 connections are important for the acquisition of new motor skills, and CB-dependent changes in M1 neuroplasticity are one way by which CB may influence motor output.

While this literature demonstrates the capacity of CB to influence M1 plasticity in a functionally relevant way, it remains unclear how this influence is mediated. In particular, the circuits within M1 that are targeted by CB are unknown. Given that previous research using TMS has shown that the activity of specific intracortical motor circuits relates to the acquisition of different motor skills [15], identification of the M1 circuitry that is affected by CB projections may allow the targeted modification of skill acquisition. Interestingly, growing evidence suggests that late indirect (I) wave inputs on to corticospinal neurons, which represent important predictors of neuroplasticity and motor learning [15-17], may be specifically modified by changes in CB excitability. For example, application of transcranial direct current stimulation (tDCS; a NIBS paradigm that induces neuroplastic changes in brain excitability) over CB specifically modulates paired-pulse TMS measures of late I-wave excitability [18]. In addition, the effects of CB tDCS on single-pulse TMS measures of M1 excitability are only apparent when stimulation is applied with an anterior-posterior current, which specifically activates late I-wave circuits [19]. Also, changes in late I-wave circuits following motor training were observed following a CB-dependent motor task, but were absent following a task with minimal CB involvement [15].

Based on this previous literature, it appears likely that CB projections to M1 influence activity within the late I-wave circuitry. However, the nature of this influence, particularly in relation to the plasticity of these circuits, remains unclear. The aim of this exploratory study is therefore to assess how changes in CB activity influence the excitability and plasticity of I-wave generating circuits in M1. To achieve this, CB excitability will be downregulated using cathodal tDCS, whereas plasticity targeting the early and late I-wave circuits will be concurrently induced using I-wave periodicity TMS (iTMS;[20, 21]).

Methods

Sample size and participants

While the effects of CB tDCS on iTMS have not been previously investigated, the study by Ates and colleagues [18] investigated the influence of CB tDCS on the excitability of the I-wave generating circuits. Consequently, sample size calculations based on this study are sufficient to demonstrate the effects of activation within the pathway of interest (i.e., cerebellar projections to I-wave circuits of M1). Examination of the findings reported by Ates and colleagues revealed that changes in short-interval intracortical facilitation (SICF; paired-pulse TMS protocol indexing I-wave excitability; [22, 23]) due to CB tDCS had an effect size of 0.67. Based on the results of an a priori power analysis utilising this effect size, with α = 0.05 and 1-β = 0.9, we will therefore recruit 21 individuals (half female) to participate in the proposed experiment.

All participants will be recruited via advertisements placed on notice boards within the University of Adelaide, in addition to on social media platforms. Exclusion criteria will include a history of psychiatric or neurological disease, current use of medications that effect the central nervous system, or left handedness. Suitability to receive TMS will be assessed using a standard screening questionnaire [24]. The experiment will be conducted in accordance with the Declaration of Helsinki, and has been approved by the University of Adelaide Human Research Ethics Committee (approval H-2019-252). Written, informed consent will be provided prior to participation. Upon completion of the study, all data will be made fully available via hosting on the open science framework repository (https://www.cos.io/osf).

Experimental arrangement

All participants will attend the laboratory for three separate sessions, with a washout period of at least 1 week between sessions. While the experimental protocol applied within each session will be the same, the ISI used for iTMS will vary between sessions (see below & Fig 1). Furthermore, the order in which each iTMS interval is applied will be randomised between participants. As diurnal variations in cortisol can influence the neuroplastic response to TMS, all plasticity interventions will be applied after 11 am and at approximately the same time of day within each participant.

Fig 1

Experimental protocol.

RMT, resting motor threshold; MEP, standard MEP of ~ 1 mV at baseline; CBI, cerebellar-brain inhibition; SICF, short-interval intracortical facilitation; MEP, standard MEP of ~ 0.5 mV at baseline with PA orientation; MEP, standard MEP of ~ 0.5 mV at baseline with AP orientation; tDCS, transcranial direct current stimulation applied to the cerebellum; iTMS, I-wave periodicity repetitive transcranial magnetic stimulation.

During each experimental session, the participant will be seated in a comfortable chair with their hand resting on a table in front of them. Surface electromyography (EMG) will be recorded from the first dorsal interosseous (FDI) of the right hand using two Ag-AgCl electrodes arranged in a belly-tendon montage on the skin above the muscle. A third electrode attached above the styloid process of the right ulnar will ground the electrodes. EMG signals will be amplified (300x) and filtered (band-pass 20Hz– 1 kHz) using a CED 1902 signal conditioner (Cambridge Electronic Design, Cambridge, UK) before being digitized at 2 kHz using CED 1401 analogue-to-digital converter and stored on a PC for offline analysis. Signal noise associated with mains power (within the 50 Hz frequency band) will also be removed using a Humbug mains noise eliminator (Quest Scientific, North Vancouver, Canada). To facilitate muscle relaxation when required, real-time EMG signals will be displayed on an oscilloscope placed in front of the participant.

Experimental procedures

Transcranial magnetic stimulation (TMS)

A figure-of-8 coil connected to two Magstim 2002 magnetic stimulators (Magstim, Dyfed, UK) via a BiStim unit will be used to apply TMS to the left M1. The coil will be held tangentially to the scalp, at an angle of 45° to the sagittal plane, with the handle pointing backwards and laterally, inducing a posterior-to-anterior (PA) current within the brain. The location producing the largest and most consistent motor evoked potential (MEP) within the relaxed FDI muscle of the right hand will be identified and marked on the scalp for reference; this target location will be closely monitored throughout the experiment. All pre- and post-intervention TMS will be applied at a rate of 0.2 Hz, with a 10% jitter between trials in order to avoid anticipation of the stimulus.

Resting motor threshold (RMT) will be defined as the stimulus intensity producing an MEP amplitude ≥ 50 μV in at least 5 out of 10 trials during relaxation of the right FDI. RMT will be assessed at the beginning of each experimental session and expressed as a percentage of maximum stimulator output (%MSO). Following assessment of RMT, the stimulus intensity producing a standard MEP amplitude of approximately 1 mV (MEP), when averaged over 20 trials, will be identified. The same intensity will then be applied 5 mins and 30 mins following the intervention in order to assess changes in corticospinal excitability.

I-wave excitability

As assessing the influence of CB modulation on I-wave excitability is the main aim of this project, changes in SICF will be the primary outcome measure. This paired-pulse TMS protocol produces MEP facilitation when conditioning and test stimuli are separated by discrete ISIs that correspond to I-wave latencies recorded from the epidural space [22]. SICF will utilise a conditioning stimulus set at 90% RMT, a test stimulus set at MEP and two ISIs of 1.5 and 4.5 ms, which correspond to the early and late MEP peaks apparent in a complete SICF curve [22, 25, 26]. Measurements of SICF will include 12 trials for each condition, at each time point.

As a secondary measure of I-wave function, TMS will be applied with different stimulus directions, which alters the interneuronal circuits contributing to the generated MEP [16, 27, 28]. When TMS is applied with a conventional (PA) current direction, the resulting MEP is thought to arise from preferential activation of early I1-waves. In contrast, when the induced current is directed from anterior-to-posterior (AP; coil handle held 180° to the PA orientation), the resulting MEP is thought to arise from preferential activation of later (I2-3) I-waves. The stimulus intensity producing an MEP of approximately 0.5 mV will be assessed for both PA (MEP) and AP (MEP) orientations at baseline. The same intensities will then be reapplied 5 mins and 30 mins after the intervention, with 20 trials applied at each time point. While the I-wave specificity of these measures is generally suggested to rely on concurrent activation of the target muscle [27], post-intervention muscle activation is also known to strongly influence neuroplasticity induction [29-31]. As the current study is primarily concerned with plasticity induction, these measures will therefore be applied in a resting muscle in order to minimize confounding effects of voluntary contraction. Given the likely independence of the intracortical circuits activated with different currents, these measures will still provide useful physiological insight.

Cerebellar-brain inhibition (CBI)

The strength of CB’s inhibitory influence on M1 will be assessed using CBI, a stimulation protocol involving a conditioning stimulus applied to CB 5 ms prior to a test stimulus applied to M1 [5]. In accordance with previous literature, CB stimulation will be applied using a double cone coil, with the center of the coil located 3 cm lateral and 1 cm inferior to the inion, along the line joining the inion and the external auditory meatus of the right ear. The coil current will be directed downward, resulting in an upward induced current. The intensity of CB stimulation will be set at 60% MSO [32], whereas M1 stimulation will be set at MEP. The dual coil configuration of this measurement will mean that each coil will be directly connected to an individual Magstim 2002 stimulator. As removing the BiStim unit will result in an increase in stimulus strength, the MEP intensity will be checked prior to baseline CBI measures, and adjusted when required. Because antidromic activation of corticospinal neurons may confound measures of CBI [33], we will ensure that the CB conditioning stimulus is at least 5% MSO below the active motor threshold for the corticospinal tract [34]. Measures of CBI will be assessed at baseline, 5 mins and 30 mins post-intervention, with 15 trials recorded for each condition at each time point.

I-wave periodicity repetitive TMS (iTMS)

In accordance with previous literature [21, 35], iTMS will consist of 180 pairs of stimuli applied every 5 s, resulting in a total intervention time of 15 mins. The same intensity will be used for both stimuli, adjusted to produce a response of ~ 1mV when applied as a pair. These stimuli will be applied using ISI’s of 1.5 (iTMS) and 4.5 ms (iTMS) in separate sessions. These parameters produce robust potentiation of MEP amplitude [20, 21, 35, 36]. A sham stimulation condition (iTMS) that is not expected to modulate corticospinal excitability will also be applied in a third session. Within this condition, we will stimulate intervals that correspond to the transition between the peaks and troughs of facilitation that are observed within a complete SICF curve, as these are not expected to induce any changes in excitability. This will include equal repetitions of 1.8, 2.3, 3.3, 3.8 and 4.7 ms ISI’s, applied randomly and with an inter-trial jitter of 10%.

Cerebellar transcranial direct current stimulation (tDCSCB)

A Soterix Medical 1 x 1 DC stimulator (Soterix Medical, New York, NY) will be used to apply tDCS to CB. Current will be applied through saline-soaked sponge electrodes (EASYpads, 5 x 7 cm), with the cathode positioned over the same location used for CB TMS (i.e., 3 cm lateral and 1 cm inferior to inion, contralateral to M1 TMS) and anode positioned on the skin above the right Buccinator muscle [4, 13, 37]. Stimulation will be applied at an intensity of 2 mA for 15 mins [4, 13, 37], coincident with the application of iTMS to M1. Onset and offset of stimulation will be ramped over a 30 s period prior to and following iTMS application.

Data analysis

Analysis of EMG data will be completed manually via visual inspection of offline recordings. For measures in resting muscle, any trials with EMG activity exceeding 25 μV in the 100 ms prior to stimulus application will be excluded from analysis. All MEPs will be measured peak-to-peak and expressed in mV. Measures of CBI will be quantified by expressing the amplitude of individual trials produced by paired-pulse stimulation as a percentage of the mean response produced by single-pulse stimulation within the same block. For baseline measures of SICF, individual trials produced by paired-pulse stimulation will be expressed as a percentage of the mean response produced by single-pulse stimulation within the same block. However, for post-intervention responses, previous work has suggested that increased facilitation following iTMS correlates with the increased response to single pulse stimulation, and that this relationship cancels the effects of iTMS on SICF if the post-intervention single-pulse MEPs are used to normalise post-intervention SICF values [21]. Consequently, prior to normalization of post-intervention SICF values, we will use linear regression analysis to assess the relationship between post-intervention single and paired-pulse responses. If the relationship is significant, individual post-intervention SICF trials will be expressed relative to the mean pre-intervention single-pulse MEP [21]. However, if the relationship is not significant, post-intervention SICF measures will be expressed relative to the single-pulse response recorded in the same block. For all TMS measures, effects of the intervention will be quantified by expressing the post-intervention responses (normalised to the relevant single-pulse response for CBI and SICF) as a percentage of the pre-intervention responses.

Statistical analysis

Kolmogorov-Smirnov tests will be applied to assess data distribution prior to statistical analysis, with log transformation applied when deviations from normal are indicated. Given that all data of interest will involve repeated-measures, linear mixed-model analysis with repeated-measures (LMM) will be used to perform all statistical comparisons. Each model will include single trial data, and previously reported methods [38] will be used to justify inclusion of random participant intercepts and/or slopes, and to optimize and assess model fit. To ensure measures are comparable between sessions, effects of iTMS session (iTMS, iTMS & iTMS) on baseline measures of MEP, MEP, MEP and CBI, in addition to responses recorded at the start of the iTMS intervention, will be investigated using one-factor LMM, with each measurement investigated in a separate model. Furthermore, effects of iTMS session and ISI (1.5 & 4.5 ms) on baseline SICF will be assessed using two-factor LMM.

Changes in excitability during the intervention will be assessed by comparing values averaged over the first, middle and last 12 stimuli of the iTMS block between iTMS sessions. Changes in corticospinal excitability following the intervention will be investigated by assessing the effects of iTMS session and time (Post 5, Post 30) on baseline-normalised MEP values. Changes in coil-orientation dependent measures of I-wave excitability following the intervention will be investigated by assessing effects of iTMS session, time and coil orientation (MEP, MEP) on baseline-normalised values. Changes in SICF measures of I-wave excitability following the intervention will be investigated by assessing effects of iTMS session, time and ISI on baseline-normalised values. Changes in CBI following the intervention will be investigated by assessing the effects of iTMS session and time on baseline-normalised CBI values. For all models, investigation of main effects and interactions will be performed using custom contrasts with Bonferroni correction, and significance will be set at P < 0.05. Data for all models will be presented as the estimated marginal means, whereas pairwise comparisons will be presented as the estimated mean difference (EMD) and 95% confidence interval (95%CI) for the estimate, providing a non-standardised measure of effect size.

Regression analyses will be used to investigate interactions between variables. Specifically, changes in CBI due to the intervention will be regressed against changes in measures of corticospinal and intracortical function in order to assess if alterations within the CB-M1 pathway contribute to plasticity effects. In addition, changes in intracortical function due to the intervention will be regressed against changes in corticospinal function in an attempt to identify if generalised changes in excitability are driven by alterations within specific circuits.

22 May 2020

PONE-D-20-08985

Characterising the influence of cerebellum on the neuroplastic modulation of intracortical motor circuits.

PLOS ONE

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Reviewer #1: The authors proposed a study to investigate how cerebellar inputs affect the motor cortical circuits producing indirect waves which are related to the cortical plasticity. The topic is interesting and the proposed study is worth doing.

1, My first major concern is that it is not clear whether this is a hypothesis driven or an exploratory protocol.

2, It seems that the central hypothesis is that cerebellar inputs change the production of later indirect wave. Related to this central hypothesis, I can not find a null hypothesis established by the authors. In particular, the last sentence of the introduction is difficult to understand.

3, There are a lot of measurements in the protocol. According to the null hypothesis, what is the primary outcome measure?

4, Sample size calculation is based on a previous study testing changes in short interval intracortical facilitation after anodal transcranial direct current stimulation. I do not understand the rationale behind this. Is this facilitation your primary outcome measure? May your study with cathodal stimulation have a different effect size from that with anodal stimulation?

Although this is a proposal and no data collection has been performed, some technical issues may be described in further details to ensure a publication.

5, Different stimulus intensities for the same cerebellar stimulation were described in the method. The authors may want to follow the detailed methods of previous studies (already cited in the manuscript) and define the intensity clearly. I agree with the opinion that the intensity should be tolerable. But low intensity stimulation will not produce cerebellar inhibition.

6, I do not understand how the sham stimulation may be performed in a way that no more than 2 repeats of any of 30 conditions are arranged in an intervention with 180 pairs of stimuli. With my calculation each condition will be repeated for 6 times in average.

7, I can not find where cerebellar direct current stimulation will be located.

8, Will the motor evoked potentials with posterior-anterior and anterior-posterior currents be measured in an active muscle? It may also need a little background introduction how such measurements are related to the later indirect wave recruitment.

9, What are the variables for regression analysis?

Reviewer #2: This is an interesting proposal and methodologically sound. I have listed a few recommendations / or points for consideration below. There may be no right answer to some of these suggestions.

I-wave excitability: “The stimulus intensity producing an MEP of approximately 0.5 mV in an active muscle will be assessed for both PA (MEPPA) an AP (MEPAP) orientations at baseline. A lower amplitude will be targeted in order to reduce stimulus intensity, and minimize concurrent recruitment of multiple I-waves.” I guess some researchers would consider contraction necessary while others might not. In my view, given the potential for contraction to interfere with the effects of some plasticity paradigms, I would personally avoid contraction pre- and post- ITMS/cTDCS. If it appears that equivalent results could be obtained and justified without contraction, then I would recommend this.

For the main SICF measures, one could consider aiming to reduce the number of pulses as in theory these could interact with the intervention.

ITMS ISI could be individualised for the 1.5 and 4.5ms ISI. It appears that a full ISI curve will be collected and SICF ISI individualised anyway. Some studies have found stronger effects, at least for ITMS1.5, when the ISI is individualised.

I think some previous ITMS studies used lower stimulus intensities, whereby e.g. “Stimulus intensity was the same for each pulse in the pair, and set so that paired stimulation generated a MEP of ~1 mV peak–peak amplitude.” The intensity required to achieve this may be less than that proposed here – I am not sure whether this is important. The authors could consider recording during ITMS and using this as a measure of SICF, provided a relevant baseline single pulse measure of equal intensity is available.

The sham condition could perhaps be designed such that it has an equal number of ISIs which fall at peaks or troughs. For example, if 2/3 of pulses happened to fall at “facilitatory” SICF intervals, it is conceivable that a somewhat “non-specific” facilitatory effect could occur. Also, I guess it is unclear whether this is relevant, but at ISIs >5ms, the MEP may begin to separate into two components – this may no longer involve what we typically think of as SICF.

Cathodal tDCS: In motor cortex, 2mA cathodal tDCS can elicit excitatory changes and differ to the depressive effects of 1mA tDCS. I am not sure whether this is the case for the Cerebellum, perhaps not.

With regard to post-intervention measures, the authors propose “Changes in SICF measures of I-wave excitability following the intervention will be investigated by assessing effects of iTMS session, time and ISI on baseline-normalised values.” I think this would be consistent with the study by Cash et al, 2009, where for the post-ITMS SICF curve “A similar analysis was performed on the post-intervention data, with the IPI data expressed as a percentage of the pre-intervention mean control value for each subject.” The authors of that paper explained this as follows “The correlation between the mean increase in the amplitude of the facilitation curve and the increase in the single-pulse MEP amplitude as a result of the intervention would mean that adjusting stimulus intensity based on the change in single pulse MEP amplitude would have obscured a change in the amplitude of the facilitation curves. Likewise, using the post-intervention single-pulse MEP as the control value for the facilitation curve post-intervention cancelled the increase in the I-wave peaks, consistent with the correlation between these measures.” I think the present authors are taking this point into account, but as it is a subtle point, I thought it might be worth flagging.

To demonstrate functional relevance, it could be interesting to include a behavioural measure or task. The authors mention relevance to motor learning. Perhaps a task could be performed early on to avoid interference with ITMS and again towards the end of the session to avoid and depotentiation etc. But this might miss the window of maximum plastic effects or increase the measurement of confounding fatigue during the course of the session. Also, perhaps difficult to find a task that works across repeated sessions.

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Reviewer #1: No

Reviewer #2: Yes: Robin Cash

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26 May 2020

Summary

This document represent a point-by-point response to the concerns raised by two reviewers. As a result of these concerns, we have made substantial changes to the manuscript. In particular, clarification of the nature (exploratory vs hypothesis driven) and methodological approach of the proposed research has been provided for reviewer 1, whereas we have modified several aspects of the proposed methodology in response to the comments from reviewer 2. We thank the reviewers for their input and believe that these changes have substantially improved the quality of the manuscript.

Response to Reviewer 1

1. My first major concern is that it is not clear whether this is a hypothesis driven or an exploratory protocol.

Response:

We agree that this element of the project is poorly defined; although the submitted manuscript included specific hypotheses about the influence of the cerebellum on I-wave circuits, the project is intended to assess a number of mechanisms associated with this potential interaction. We therefore feel that it must be considered to be primarily exploratory in nature. Consequently, we have removed the specific hypotheses from the revised manuscript and reworded the final paragraph of the introduction to clarify the exploratory nature of the research.

2. It seems that the central hypothesis is that cerebellar inputs change the production of later indirect wave. Related to this central hypothesis, I cannot find a null hypothesis established by the authors. In particular, the last sentence of the introduction is difficult to understand.

Response:

While we appreciate the point raised by the reviewer, the research is exploratory in nature and we therefore do not believe that inclusion of an alternative hypothesis is warranted.

3. There are a lot of measurements in the protocol. According to the null hypothesis, what is the primary outcome measure?

Response:

As the main aim of the proposed research is to assess if cerebellar modulation influences neuroplasticity of the I-wave generating circuits, specific measures of I-wave excitability are the main outcome of interest. In the context of our study, changes in SICF due to the iTMS intervention are therefore the primary outcome measure, and we have clarified this within the revised manuscript (page 6). However, given the exploratory nature of this research, this has not been framed in the context of a null hypothesis (see our response to the reviewer’s second concern).

4. Sample size calculation is based on a previous study testing changes in short interval intracortical facilitation after anodal transcranial direct current stimulation. I do not understand the rationale behind this. Is this facilitation your primary outcome measure? May your study with cathodal stimulation have a different effect size from that with anodal stimulation?

Response:

As suggested by the reviewer, SICF is the primary outcome measure for the proposed study. This has been clarified in response to the reviewer’s third concern. Unfortunately, the exploratory nature of this research means that no previous work has investigated the influence of cerebellar tDCS (cathodal or anodal) on changes in SICF due to iTMS, and we are therefore unable able to base sample size estimates on an ideal dataset. However, the study by Ates et al. (2018) utilises cerebellar tDCS to modify the excitability of the I-wave generating circuits. We therefore believe that sample size calculations based on the effects of this study are sufficient to demonstrate the effects of activation within the pathway of interest (i.e., cerebellar projections to I-wave circuits of M1). While the Ates study does not inform sample sizes required to detect the response to iTMS, the proposed number of participants exceeds the numbers that have been included within previous studies reporting significant effects of this paradigm (more than doubling the sample in some instances). Consequently, the proposed sample size should be sufficient to detect effects of iTMS and effects of cerebellar modulation on SICF. We have clarified our use of the Ates study for sample size calculations within the revised manuscript (page 4).

5. Different stimulus intensities for the same cerebellar stimulation were described in the method. The authors may want to follow the detailed methods of previous studies (already cited in the manuscript) and define the intensity clearly. I agree with the opinion that the intensity should be tolerable. But low intensity stimulation will not produce cerebellar inhibition.

Response:

We agree with the reviewer’s suggestion, and have therefore modified the revised manuscript to state that a conditioning intensity of 60% MSO will be applied for measures of CBI. This intensity was chosen based on previous work characterising the recruitment of CBI, which showed that 60% MSO produces significant inhibition, but that intensities greater than this do not produce any more inhibition (Fernandez et al., 2018). Consequently, this level of stimulation finds a balance between achieving a significant level of CBI while maintaining tolerability for the participant.

6. I do not understand how the sham stimulation may be performed in a way that no more than 2 repeats of any of 30 conditions are arranged in an intervention with 180 pairs of stimuli. With my calculation each condition will be repeated for 6 times in average.

Response:

Thanks to the reviewer for identifying this typographical error. As suggested, it should have read no more than 6 repeats. However, in response to reviewer 2’s fifth concern, an alternative approach to the sham stimulation has been adopted within the revised manuscript.

7. I cannot find where cerebellar direct current stimulation will be located.

Response:

Apologies to the reviewer for omitting this information. The cathode for cerebellar tDCS will be centred over the same location that is used for cerebellar TMS, 3 cm lateral and 1 cm inferior to the inion, on the line joining the inion and external auditory meatus of the right ear. This has been clarified within the revised manuscript (page 8)

8. Will the motor evoked potentials with posterior-anterior and anterior-posterior currents be measured in an active muscle? It may also need a little background introduction how such measurements are related to the later indirect wave recruitment.

Response:

The original protocol included PA/AP MEPs in an active muscle, as this approach minimises the need for multiple I-wave volleys to overcome the spinal motoneurone activation threshold, producing responses that are more likely to be reflective of inputs from single I-waves. However, in order to respond to reviewer 2’s first concern, these measures will now be recorded in a resting muscle.

9. What are the variables for regression analysis?

Response:

Changes in CBI due to the intervention will be regressed against changes in measures of corticospinal (MEP1mV) and intracortical (SICF, MEPPA/AP) function in order to assess if alterations within the CB-M1 pathway contribute to plasticity effects. In addition, changes in intracortical function due to the intervention will be regressed against changes in corticospinal function in an attempt to identify if generalised changes in excitability are driven by changes in specific circuits. We have included this information in the revised manuscript (page 10/11).

Also, post-intervention changes in the response to single and paired stimulation will be correlated against each other in order to select the most appropriate normalisation approach (see the response to reviewer 2’s seventh concern).

Response to Reviewer 2

1. I-wave excitability: “The stimulus intensity producing an MEP of approximately 0.5 mV in an active muscle will be assessed for both PA (MEPPA) an AP (MEPAP) orientations at baseline. A lower amplitude will be targeted in order to reduce stimulus intensity, and minimize concurrent recruitment of multiple I-waves.” I guess some researchers would consider contraction necessary while others might not. In my view, given the potential for contraction to interfere with the effects of some plasticity paradigms, I would personally avoid contraction pre- and post- ITMS/cTDCS. If it appears that equivalent results could be obtained and justified without contraction, then I would recommend this.

Response:

Thanks to the reviewer for their comment, it’s a very good point. Generally, we view muscle activation and low stimulus intensities as a necessity for ensuring measures that are I-wave selective when changing stimulus direction (Day et al., 1989). However, the main aim of the study is to assess the plasticity response, and the protocol must therefore be optimised to achieve this. We agree that muscle activation could interfere with any neuroplastic effects, and we will therefore apply directional measures in a resting muscle. Given the potential independence of the I-wave circuits activated with different current directions, we believe that these measures will still be of value for identifying contributions from different intracortical circuits. The revised manuscript has been updated to reflect these changes (page 7).

2. For the main SICF measures, one could consider aiming to reduce the number of pulses as in theory these could interact with the intervention.

Response:

We agree with the point raised by the reviewer and have therefore revised the protocol to include 12 trials per condition. However, given the variability of these measures, we would prefer not to reduce this number any further.

3. ITMS ISI could be individualised for the 1.5 and 4.5ms ISI. It appears that a full ISI curve will be collected and SICF ISI individualised anyway. Some studies have found stronger effects, at least for ITMS1.5, when the ISI is individualised.

Response:

Apologies for the confusion, but we do not intend to record a full SICF curve. Only standard ISIs associated with the first (1.5 ms) and third (4.5 ms) peak will be tested. While we agree that a full curve would be interesting, and that individualised iTMS would be ideal, time limitations do not allow us to include these measures. If cerebellar modulation is able to influence iTMS effects, these are factors we intend to pursue further in future studies.

4. I think some previous ITMS studies used lower stimulus intensities, whereby e.g. “Stimulus intensity was the same for each pulse in the pair, and set so that paired stimulation generated a MEP of ~1 mV peak–peak amplitude.” The intensity required to achieve this may be less than that proposed here – I am not sure whether this is important. The authors could consider recording during ITMS and using this as a measure of SICF, provided a relevant baseline single pulse measure of equal intensity is available.

Response:

Review of the iTMS literature indeed suggests that a large number of previous studies have employed lower intensity stimuli during the intervention. In line with Cash et al. (2009), we will therefore use paired stimuli producing an MEP of ~ 1 mV during the intervention. We intend to record responses during iTMS in order to track changes in excitability during the intervention. However, previous work suggests that single pulse stimulation applied at the intensity producing a response of ~1 mV as a pair does not produce an MEP (Thickbroom et al., 2006). It therefore seems likely that the MEP response to single-pulse stimulation applied at the intervention intensity will be too small for normalisation purposes. Consequently, we would prefer to retain the pre- and post-intervention SICF parameters as they were originally proposed. In contrast, changes in excitability during the intervention will be quantified by comparing the first, middle and last 12 stimuli. These alterations have been included within the revised manuscript (page 10).

5. The sham condition could perhaps be designed such that it has an equal number of ISIs which fall at peaks or troughs. For example, if 2/3 of pulses happened to fall at “facilitatory” SICF intervals, it is conceivable that a somewhat “non-specific” facilitatory effect could occur. Also, I guess it is unclear whether this is relevant, but at ISIs >5ms, the MEP may begin to separate into two components – this may no longer involve what we typically think of as SICF.

Response:

The reviewer raises a good point that we had not considered, and we have therefore modified the sham stimulation to counterbalance the number of facilitatory and inhibitory ISI’s. Facilitatory ISI’s of 1.5, 3 and 4.5 ms, and inhibitory ISI’s of 2 and 3.5 ms will be used during sham stimulation. These will be randomly applied, with 30 repeats for each facilitatory ISI and 45 repeats for each inhibitory ISI. These changes have been included in the revised manuscript (page 8).

6. Cathodal tDCS: In motor cortex, 2mA cathodal tDCS can elicit excitatory changes and differ to the depressive effects of 1mA tDCS. I am not sure whether this is the case for the Cerebellum, perhaps not.

Response:

Previous work suggests that disinhibitory effects of cathodal tDCS over cerebellum actually require a higher intensity of 2 mA (Galea et al., 2009; Fig 6A). While we agree that effects of tDCS on M1 have a complex relationship that is intensity-dependent, we therefore feel that this intensity is appropriate in this context.

7. With regard to post-intervention measures, the authors propose “Changes in SICF measures of I-wave excitability following the intervention will be investigated by assessing effects of iTMS session, time and ISI on baseline-normalised values.” I think this would be consistent with the study by Cash et al, 2009, where for the post-ITMS SICF curve “A similar analysis was performed on the post-intervention data, with the IPI data expressed as a percentage of the pre-intervention mean control value for each subject.” The authors of that paper explained this as follows “The correlation between the mean increase in the amplitude of the facilitation curve and the increase in the single-pulse MEP amplitude as a result of the intervention would mean that adjusting stimulus intensity based on the change in single pulse MEP amplitude would have obscured a change in the amplitude of the facilitation curves. Likewise, using the post-intervention single-pulse MEP as the control value for the facilitation curve post-intervention cancelled the increase in the I-wave peaks, consistent with the correlation between these measures.” I think the present authors are taking this point into account, but as it is a subtle point, I thought it might be worth flagging.

Response:

Thanks to the reviewer for bringing this subtle point to our attention. We had originally planned to quantify paired-pulse facilitation relative to the single-pulse response recorded at the same time point, and to then normalise these values to baseline SICF. However, we will now assess the relationship between single and paired-pulse responses post-intervention, and quantify facilitation based on the pre-intervention single-pulse response if there is a significant correlation. If the correlation proves to be non-significant, we believe the original approach will be more appropriate. These changes have been included in the revised manuscript (page 9).

8. To demonstrate functional relevance, it could be interesting to include a behavioural measure or task. The authors mention relevance to motor learning. Perhaps a task could be performed early on to avoid interference with ITMS and again towards the end of the session to avoid and depotentiation etc. But this might miss the window of maximum plastic effects or increase the measurement of confounding fatigue during the course of the session. Also, perhaps difficult to find a task that works across repeated sessions.

Response:

We agree that the inclusion of a behavioural measure would be very interesting. However, as suggested in response to the reviewer’s first concern, the main interest of this study is to assess the potential influence of cerebellum on plasticity mechanisms of M1, and we therefore need to optimise the protocol to achieve this. While it seems possible that distancing task performance in time may reduce its influence on the plasticity response, we cannot be sure to what extent this is true. Furthermore, as suggested by the reviewer, it is quite likely that this delay will reduce the likelihood of identifying any functional effects of the intervention. Finally, time constraints make it difficult to include additional measures without adding sessions, which we would prefer to avoid. Taken together, it seems that properly investigating the question of functional relevance will require a purpose-built experimental protocol. If the proposed intervention demonstrates physiological benefit, this is an avenue we will pursue further.

References

Ates MP, Alaydin HC & Cengiz B. (2018). The effect of the anodal transcranial direct current stimulation over the cerebellum on the motor cortex excitability. Brain Res Bull 140, 114-119.

Cash R, Benwell N, Murray K, Mastaglia F & Thickbroom G. (2009). Neuromodulation by paired-pulse TMS at an I-wave interval facilitates multiple I-waves. Exp Brain Res 193, 1-7.

Day BL, Dressler D, Denoordhout AM, Marsden CD, Nakashima K, Rothwell JC & Thompson PD. (1989). Electric and magnetic stimulation of human motor cortex - surface EMG and single motor unit responses. J Physiol-London 412, 449-473.

Fernandez L, Major BP, Teo W-P, Byrne LK & Enticott PG. (2018). The impact of stimulation intensity and coil type on reliability and tolerability of cerebellar brain inhibition (CBI) via dual-coil TMS. Cerebellum 17, 540-549.

Galea JM, Jayaram G, Ajagbe L & Celnik P. (2009). Modulation of cerebellar excitability by polarity-specific noninvasive direct current stimulation. J Neurosci 29, 9115-9122.

Thickbroom GW, Byrnes ML, Edwards DJ & Mastaglia FL. (2006). Repetitive paired-pulse TMS at I-wave periodicity markedly increases corticospinal excitability: a new technique for modulating synaptic plasticity. Clin Neurophysiol 117, 61-66.

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17 Jun 2020

PONE-D-20-08985R1

Characterising the influence of cerebellum on the neuroplastic modulation of intracortical motor circuits.

PLOS ONE

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Reviewer 2 raised a point to be considered regarding sham stimulation.

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Reviewers' comments:

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Comments to the Author

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Reviewer #2: Yes

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Reviewer #2: The protocol is well thought out and the authors have done a great and timely job of responding.

I have one minor comment with regard to sham stimulation - depending on the intensities used for SICF/ITMS, paired pulse ISIs could be facilitatory, rather than inhibitory. I think this point might be illustrated in the figures of Ilic and colleagues (2002). E.g. I think it is even possible to have facilitation at ISI 2ms, depending on the intensity. Personally I would probably avoid including ISI 1.5ms (or any in the range of 1.1-1.7ms) in the sham condition and try to select ISIs that are intermediate to peaks and troughs - ISIs that would be anticipated to show little facilitation or inhibition at the intensities employed. Ultimately, there is probably no ideal way to circumvent this issue but I raise it for the author's consideration.

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Reviewer #2: Yes: Robin Cash

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18 Jun 2020

Reviewer 2 comment:

I have one minor comment with regard to sham stimulation - depending on the intensities used for SICF/ITMS, paired pulse ISIs could be facilitatory, rather than inhibitory. I think this point might be illustrated in the figures of Ilic and colleagues (2002). E.g. I think it is even possible to have facilitation at ISI 2ms, depending on the intensity. Personally I would probably avoid including ISI 1.5ms (or any in the range of 1.1-1.7ms) in the sham condition and try to select ISIs that are intermediate to peaks and troughs - ISIs that would be anticipated to show little facilitation or inhibition at the intensities employed. Ultimately, there is probably no ideal way to circumvent this issue but I raise it for the author's consideration.

Response:

Thanks to the reviewer for their additional comment. As suggested, the issue of sham stimulation for the iTMS protocol represents a significant challenge. However, we appreciate the point being made, and will therefore modify the sham protocol to only use ISI’s that represents transitions between peaks and troughs of facilitation. Consequently, the sham protocol will include equal repetitions of ISI’s 1.8, 2.3, 3.3, 3.8 and 4.7 ms. These intervals were identified based on facilitation curves presented by several previous studies (Ziemann et al., 1998; Delvendahl et al., 2014; Cirillo et al., 2015; Opie et al., 2018). The revised manuscript has been updated to include these details (page 8).

Cirillo J, Calabro FJ & Perez MA. (2015). Impaired organization of paired-pulse TMS-induced I-waves after human spinal cord injury. Cereb Cortex 26, 2167-2177.

Delvendahl I, Lindemann H, Jung NH, Pechmann A, Siebner HR & Mall V. (2014). Influence of waveform and current direction on short-interval intracortical facilitation: a paired-pulse TMS study. Brain Stimul 7, 49-58.

Opie GM, Cirillo J & Semmler JG. (2018). Age‐related changes in late I‐waves influence motor cortex plasticity induction in older adults. J Physiol 596, 2597-2609.

Ziemann U, Tergau F, Wassermann EM, Wischer S, Hildebrandt J & Paulus W. (1998). Demonstration of facilitatory I wave interaction in the human motor cortex by paired transcranial magnetic stimulation. J Physiol 511, 181-190.

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29 Jun 2020

Characterising the influence of cerebellum on the neuroplastic modulation of intracortical motor circuits.

PONE-D-20-08985R2

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1 Jul 2020

PONE-D-20-08985R2

Characterising the influence of cerebellum on the neuroplastic modulation of intracortical motor circuits.

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