- Email: [email protected]
Anodal Cerebellar Direct Current Stimulation Reduces Facilitation of Propriospinal Neurons in Healthy Humans
Accepted Manuscript Title: Anodal Cerebellar Direct Current Stimulation Reduces Facilitation of Propriospinal Neurons in Healthy Humans Author: Muhammed Chothia, Sebastian Doeltgen, Lynley V Bradnam PII: DOI: Reference:
S1935-861X(16)00006-1 http://dx.doi.org/doi: 10.1016/j.brs.2016.01.002 BRS 844
To appear in:
Brain Stimulation
Received date: Revised date: Accepted date:
20-10-2015 1-1-2016 4-1-2016
Please cite this article as: Muhammed Chothia, Sebastian Doeltgen, Lynley V Bradnam, Anodal Cerebellar Direct Current Stimulation Reduces Facilitation of Propriospinal Neurons in Healthy Humans, Brain Stimulation (2016), http://dx.doi.org/doi: 10.1016/j.brs.2016.01.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title: Anodal cerebellar direct current stimulation reduces facilitation of propriospinal
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neurons in healthy humans
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Authors and affiliations
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Muhammed Chothia1, Sebastian Doeltgen2 Lynley V Bradnam1,3
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1
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Flinders University, Adelaide, South Australia, 5041
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2
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University, South Australia, 5041
Applied Brain Research Laboratory, Discipline of Physiotherapy, Faculty of Health Sciences
Discipline of Speech Pathology and Audiology, School of Health Sciences, Flinders
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NSW, Australia
Discipline of Physiotherapy, Graduate School of Health, University of Technology Sydney,
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Corresponding Author
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Lynley Bradnam, Discipline of Physiotherapy, Graduate School of Health, University of
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Technology Sydney, PO Box 123, NSW, Australia, 2007.
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Email: [email protected]
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Running Title: Cerebellar stimulation and propriospinal neurons
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Highlights
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Activity in cervical propriospinal neurons (PNs) was explored using paired TMS and peripheral nerve stimulation Anodal cerebellar DCS was used to probe cerebellar control of propriospinal activity Facilitation of PNs was reduced by cerebellar anodal DCS, with no effect on inhibition The cerebellum may contribute to upper limb coordination by modulation of PN excitability in humans
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Abstract
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Background
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Coordinated muscle synergies in the human upper limb are controlled, in part, by a neural
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distribution network located in the cervical spinal cord, known as the cervical propriospinal
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system. Studies in cats and non-human primates indicate the cerebellum is indirectly
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connected to this system via output pathways to the brainstem. Therefore, the cerebellum
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may indirectly modulate excitability of putative propriospinal neurons (PNs) in humans
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during upper limb coordination tasks.
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Objective/Hypothesis
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This study aimed to test whether anodal direct current stimulation (DCS) of the cerebellum
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modulates PNs and upper limb coordination in healthy adults. The hypothesis was that
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cerebellar anodal DCS would reduce descending facilitation of PNs and improve upper limb
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coordination.
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Methods
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Transcranial magnetic stimulation (TMS), paired with peripheral nerve stimulation probed
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activity in facilitatory and inhibitory descending projections to PNs following an established
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protocol. Coordination was tested using a pursuit rotor task performed by the non-dominant
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(ipsilateral) hand. Anodal and sham DCS were delivered over the cerebellum ipsilateral to the
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non-dominant hand in separate experimental sessions. Anodal DCS was applied to a control
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site lateral to the vertex in a third session. Twelve right-handed healthy adults participated.
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Results
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Pairing TMS with sub-threshold peripheral nerve stimulation facilitated motor evoked
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potentials at intensities just above threshold in accordance with the protocol. Anodal
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cerebellar DCS reduced facilitation without influencing inhibition, but the reduction in
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facilitation was not associated with performance of the pursuit rotor task.
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Conclusions
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The results of this study indicate dissociated indirect control over cervical PNs by the
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cerebellum in humans. Anodal DCS of the cerebellum reduced excitability in the facilitatory
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descending pathway with no effect on the inhibitory pathway to cervical PNs. The reduction
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in PN excitability is likely secondary to modulation of primary motor cortex or brainstem
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nuclei, and identifies a neuroanatomical pathway for the cerebellum to assist in coordination
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of upper limb muscle synergies in humans.
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Keywords: propriospinal; direct current stimulation; transcranial magnetic stimulation;
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cerebellum; stroke; dystonia
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Introduction
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Reaching and grasping to manipulate objects with the hand is a fundamental task in humans,
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involving fine-tuned stereotypical movements demanding precise co-contraction of proximal
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and distal musculature. Coordinated movement relies on synergistic activation of each muscle
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involved in a functional task, as well as the coordinated inhibition of their respective
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antagonists. Abnormal patterns of movement exist between proximal and distal upper limb
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muscles in neurological disorders such as chronic stroke and dystonia [1, 2], limiting useful
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function [3]. One theory for upper extremity muscle synergies are that they are formed by
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pre-motoneuronal spinal distribution networks in the upper cervical cord [4, 5], known as the
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cervical propriospinal system. Propriospinal neurons (PNs) were described in cats [6] and
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non-human primates [7, 8] and found to contribute to muscle synergies for upper limb target
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reaching by exciting α-motor neurons (αMNs) innervating agonist muscles and suppressing
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antagonist muscle activity [9]. Propriospinal neurons integrate descending cortical commands
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with afferent feedback from the upper limb, to permit immediate update of the motor
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command [6, 10, 11] (Figure 1). Both descending (efferent) and ascending (afferent) inputs
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can directly excite PNs or suppress their activity via local inhibitory interneurons [8, 10, 12]
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so that synergies are modulated according to task requirements.
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Activity in cervical PNs can be probed indirectly in humans using transcranial magnetic
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stimulation (TMS), paired with peripheral nerve stimulation. With an appropriate
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interstimulus interval (ISI), the two stimuli summate in the putative PNs [13-17]. The ISI
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optimal range is calculated to be 6-9ms, based on the afferent and efferent conduction times
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of peripheral nerves and spinal cord white matter [5]. Previous studies demonstrated TMS at
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intensities just above threshold, paired with sub-motor threshold nerve stimulation, facilitates
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motor evoked potentials (MEPs). Slightly stronger TMS intensities paired with the same sub-
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motor threshold nerve stimulation suppress MEPs, presumably by recruitment of local Page 4
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inhibitory interneurons [15]. In humans, PNs are held under tonic inhibition which is released
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during functional tasks that require synergistic activation of proximal and distal upper limb
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muscles [14, 16].
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Experiments in cats and non-human primates indicate the cerebellum is intimately involved
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in the spinal distribution network. Propriospinal neurons send ascending projections to the
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lateral reticular nucleus in the brainstem which projects in turn to the cerebellum [18, 19].
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The extensive major mossy fibre connections between the lateral reticular nucleus and the
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cerebellum permits an efferent copy of the motor command from PNs to spinal αMNs to be
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analysed by the cerebellum [20, 21]. The cerebellum can update the motor command by its
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output projections to the primary motor cortex (M1) and brainstem motor nuclei and their
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descending pathways which terminate on PNs and αMNs [8] (Figure 1). However, direct
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evidence of modulation of PN activity by the cerebellum has not yet been demonstrated in
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humans. This question may be answered with non-invasive stimulation. The cerebellum can
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be modulated by anodal direct current stimulation (DCS) in healthy humans [22, 23] but there
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are no studies that have investigated the influence of cerebellar DCS on putative PNs in
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humans.
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The objective of this study was to examine the role of the cerebellum by probing the effect of
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anodal cerebellar DCS on facilitatory and inhibitory projections to PNs in healthy humans.
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Cerebellar DSC influence on ipsilateral upper limb coordination was tested using a pursuit
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rotor task performed with the non-dominant hand [24]. The pursuit rotor task was chosen as it
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requires precise coordination of hand and arm, considered to rely, in part, on cerebellar
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function. The a priori hypothesis was that anodal cerebellar DCS would reduce PN
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excitability, because outputs from the cerebellar cortex are inhibitory and would reduce
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descending drive from M1 or brainstem nuclei to the spinal cord. We also hypothesised that
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reduced facilitation of PNs would degrade performance of the non-dominant hand on the
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pursuit rotor task, due to impaired muscle synergies and coordination.
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Methods
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Participants
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Twelve healthy adults (mean age = 29.6 yr, range = 23-57 yr, 7 males) without history of
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neurological or musculoskeletal disorder affecting the neck or upper limb participated in the
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study, following screening for contraindications to TMS by a physician. All twelve
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participants were right handed, assessed with the Flinders Handedness survey (9.83 ± 0.11)
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[25]. Informed, written consent was obtained from each individual. Ethical approval was
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provided by the Southern Adelaide Clinical Human Research Ethics Committee. The study
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was conducted in accordance with the Declaration of Helsinki.
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Experimental Design
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Participants completed three experimental sessions separated by at least 5 days.
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Neurophysiological and behavioural data were collected pre- and post-DCS at each session.
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Participants were randomly assigned to intervention order and blinded to the stimulation at
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each session. The same experimenter delivered the intervention and collected outcome
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measures and was not blinded.
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Electromyography
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Surface electromyography (EMG) was recorded from the left biceps brachii (BB) and left
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flexor carpi radialis (FCR) using disposable electrodes (30 x 20mm; Ambu, Ballerup,
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Denmark). EMG recordings were made using a belly-tendon montage. EMG from the FCR
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was only used to set the intensity for median nerve stimulation; no data from this muscle was
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collected or analysed. EMG signals were amplified (CED 1902; Cambridge Electronic
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Design (CED), Cambridge, United Kingdom), band-pass-filtered (20 –1,000 Hz) and sampled
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at 2 kHz (CED 1401).
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Transcranial Magnetic Stimulation
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Single-pulse TMS was delivered to the right M1 to elicit MEPs in the left BB with a 70mm
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figure-of-eight remote control coil and a MagStim 2002 unit (MagStim, Dyfed, Wales). The
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coil was held over the scalp at a 45o angle to induce a posterior to anterior current in the
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underlying motor cortex. Participants held a 1000g weight in their left hand with the forearm
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in full supination, the wrist in slight flexion and the elbow flexed between 90 and 120o, in
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order to co-contract the BB and FCR during TMS [13, 17]. The optimal stimulation site
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evoking the largest MEP in the left BB was located and marked on the scalp. Active motor
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threshold (AMT) was defined for the left BB as the minimum stimulus intensity that elicited
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a 100µV response, with a size, shape and latency that was consistent with an MEP, in 5 out of
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10 trials [26], while maintaining the task position. Once AMT was determined, six stimulus
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intensities were calculated in increments of 2% maximal stimulator output (MSO), starting
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from AMT -2% of MSO. Each TMS intensity was delivered in random order as a separate
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block during task performance. The blocks consisted of 16 non-conditioned MEPs (NC) and
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16 MEPs conditioned (C) by median nerve stimulation (MNS), recorded at a rate of 0.2Hz.
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Rest breaks were provided between blocks to prevent fatigue. Responses were recorded using
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Signal Software v6 (CED) and saved for offline analysis.
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Median Nerve Stimulation
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The median nerve was stimulated using square-wave pulses (1ms) delivered by a Digitimer
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DS7A constant current stimulator (Digitimer, Hertfordshire, UK) prior to TMS. Timing
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between the two stimuli was automated using Signal software (V6). A motor point pen
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electrode (Compex) was used to locate the median nerve in the cubital fossa at the elbow.
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The optimum site for MNS was detected by observation of the motor response (M-wave) on
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the computer screen. The cathode was fixed to the skin by an adhesive electrode (Ambu,
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Ballerup, Denmark) over the optimal stimulation point for the median nerve at the elbow. The
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cathode was located proximal, and the anode distal, in relation to the spinal cord. Motor
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threshold was defined as the minimum current intensity to induce an M-wave response while
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maintaining the task position. The intensity for MNS was set to 0.8 of motor threshold, to
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preferentially stimulate group I sensory afferents [15]. To determine the optimum ISI
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between MNS and TMS for each individual, detailed exploration of different ISIs between 6-
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9 ms was undertaken using TMS intensities of AMT +2% to +4% MSO in the first
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experimental session. The ISI that generated maximum facilitation of the non-conditioned
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MEP was identified as the optimal ISI and used for all three conditions for that individual.
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The range of 6-9 ms was determined in previous studies [13, 16, 17].
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Direct Current Stimulation
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Direct current stimulation was delivered using a TCT stimulator (TCT Research, Hong Kong)
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with a constant current of 2 mA, for 15 minutes using two rubber electrodes encased in
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saline-soaked sponges. The anode was 16cm2 and the cathode was 24cm2, producing a
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current density under the anode of 1.25A/m2. The anode was positioned over the left lateral
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cerebellum, 3cm lateral to the external occipital protuberance. The cathode was placed over
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the left buccinator [23]. The sham condition used the identical electrode configuration,
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ramping the current up to 2mA and back down to 0mA over 30s [27]. To confirm any effect
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on PNs resulted from cerebellar anodal DCS, an active control condition, where the anode
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was positioned over the left parietal lobe 3cm lateral from the mid-central scalp (Cz) and the
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cathode on the left buccinator, was included. Participants sat quietly throughout the
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intervention and for 5 minutes afterwards.
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Rotor pursuit task
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Participants were seated at a desk in a standardised position with a tablet (iPad Apple Pty
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Ltd) directly in front of them, laid flat in portrait orientation. Participants were instructed to
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follow a moving cursor on the tablet screen with a stylus held in their left (non-dominant)
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hand. The cursor followed a circular path, 6.25cm in radius (Rotor Test, Stroke Science,
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University of Auckland) at a speed of 21 revolutions per minute (RPM). One practice trial of
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7 revolutions (21 RPM) was undertaken at the beginning of each experiment. After that, five
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trials of 7 revolutions (21 RPM) were completed with a 15 second rest between trials, before
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and after the DCS intervention.
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Data Analysis
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Electromyography traces from the left BB were rectified and the MEPAREA calculated between
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the onset and offset latencies using the same window for each individual, optimised across
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the three conditions. The MEPAREA was averaged for each block and expressed as a ratio of
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conditioned to non-conditioned (C/NC) responses. The facilitation element (FAC) was
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determined by a ratio greater than 1 and the following inhibition element (INH) by a ratio less
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than 1. The blocks that produced the greatest degree of FAC and INH for each participant
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were identified and the data averaged for each experimental condition. Traces from a
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representative subject are provided in Figure 2. The difference in FAC and INH elements pre
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to post DCS was calculated (Δ = post DCS – pre DCS) for each individual and averaged [17].
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Root mean square EMG (rmsEMG) was calculated between 100 and 10ms prior to the
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stimulus artefact for all MEP data.
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For the pursuit rotor task the time for which the stylus was correctly in contact with the
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moving cursor (total contact time) was recorded from each of the five individual trials in a
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trial block. The first trial of each block was used to normalise the four subsequent trials, in
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order to account for variations in task performance between individuals. The five normalised
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trials were then plotted against time and subjected to linear regression analysis. The slope of
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the linear line of best fit was calculated to indicate task performance over the five trials.
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Group averages were calculated for each condition and time point for statistical analysis. The
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difference in total contact time over the five trials before and after DCS was calculated as
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ΔSlope = post-DCS − pre-DCS.
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Statistical Analysis
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Ratios, non-conditioned MEPs and rmsEMG were tested for normality using the Shapiro-
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Wilks test. Where data did not meet assumptions of normality, data was log10 transformed
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and statistical analysis was performed on the logarithmic transformed data. For pre-
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intervention ratios, a one sample t-test was used to determine if FAC and INH were present at
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baseline, and paired sample t-tests compared baseline consistency across the three conditions.
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MEP ratios and non-conditioned MEPs were analysed using separate 3 CONDITION (Real,
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Control, Sham), x 2 ELEMENT (FAC, INH) x 2 TIME (pre, post) repeated measures ANOVA
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(rmANOVA). To compare effects of DCS across conditions, ΔFAC and ΔINH were tested
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using a 3 CONDITION (Real, Control, Sham) x 2 ELEMENT (ΔFAC, ΔINH) rmANOVA.
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Finally, root mean square EMG (rmsEMG) was analysed using a separate 3 CONDITION (Real,
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Control, Sham) x 2 MEP (Cond, NC) x 2 TIME (pre, post) rmANOVA for each ELEMENT. The
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difference in slope value for the pursuit rotor task pre to post DCS was first analysed using
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the non-parametric Wilcoxon signed-rank test, separately for each condition. Second, ΔSlope
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was analysed using a 3 CONDITION (Real, Control, Sham) rmANOVA. Spearman’s
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correlation was used to test for relationships between TMS parameters demonstrating
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significant change and task performance. The Greenhouse Geisser correction was utilised
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where data did not meet conditions of sphericity. Post hoc paired t-tests were used to explore
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significant main effects or interactions. Multiple tests were adjusted for significance using a
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modified Bonferroni test [28]. Data are expressed as mean ± SE.
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Results
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One sample t-tests revealed FAC and INH at baseline for all conditions (all P < 0.01), apart
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from the INH element in the Sham condition (P = 0.22). Paired sample t-tests revealed no
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difference for CONDITION or ELEMENT pre-stimulation (all P > 0.11) (Table 1). The
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rmANOVA revealed a main effect of ELEMENT (F1,11 = 116.56, P < 0.0001) and CONDITION
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(F2,22 = 3.46, P = 0.049), a CONDITION by TIME interaction (F2,22 = 5.88, P = 0.014) and a TIME
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by ELEMENT interaction (F1,11 = 21.10, P = 0.001). There was a strong trend for a CONDITION
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by ELEMENT by TIME interaction (P = 0.05). There was no main effect for TIME (P = 0.82) or
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an interaction between CONDITION and ELEMENT (P = 0.54) (Figure 3A). To assess the
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intervention effect, post hoc, paired sample t-tests revealed a reduction across TIME for the
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FAC ELEMENT following anodal cerebellar DCS (P < 0.001) but not for Sham and control
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DCS (both P > 0.33) (Figure 3B). For the INH ELEMENT, there was a reduction following
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control DCS (P = 0.015) but not for anodal cerebellar DCS (P = 0.1) or sham DCS (P = 0.52)
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(Table 1).
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The rmANOVA for ΔFAC and ΔINH revealed a main effect of ELEMENT (F1,11 = 21.78, P =
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0.001) and CONDITION (F2,22 = 5.86, P = 0.014), but no interaction (P = 0.07). The reduction
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in facilitation represented by ΔFAC after anodal cerebellar DCS differed from sham (P =
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0.001) and control (P = 0.009) conditions (Figure 3C). There was no difference for ∆INH
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between the three conditions (all P > 0.19). For the NC MEPs, there was a main effect of
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ELEMENT
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to evoke INH were larger (4.51 +/- 0.47 V.ms) than NC MEPs at the lower TMS intensity to
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evoke FAC (2.54 +/- 0. 34 V.ms). There were no other main effects or any interactions (all P
(F1,11 = 95.33, P < 0.0001). As expected, NC MEPs at higher TMS intensities used
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> 0.07) (Table 2). Data for the prestimulus rmsEMG were log10 transformed prior to analysis
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and the rmANOVA found prestimulus rmsEMG was consistent across condition and time for
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both FAC and INH components (both P > 0.11) (Table 2). For the pursuit rotor task, the
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Wilcoxon signed-rank tests revealed no change in slope after stimulation for all conditions
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(all Z < -0.94, all P > 0.1). For ΔSlope, there was no difference between conditions (P = 0.07)
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although observation of the data indicates slope was increased following anodal cerebellar
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DCS and decreased following sham cerebellar DCS (Figure 3D). A Spearman's rank-order
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correlation revealed there was no relationship between ΔFAC and ΔSlope (rs = 0.469, P =
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0.124) in the anodal cerebellar DCS condition.
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Discussion
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We investigated if anodal cerebellar DCS influenced descending facilitatory and inhibitory
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drive to cervical PNs in healthy humans and whether this was related to performance of an
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upper limb coordination task. The major new finding was that anodal cerebellar DCS reduced
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activity (or disfacilitation) in excitatory descending pathways to PNs, without influencing
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inhibitory pathways. Neither sham cerebellar DCS, nor an active control condition where the
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anode was placed lateral to the midline at the vertex, altered activity in either descending
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pathway. These findings suggest that anodal cerebellar DCS indirectly influenced PN
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excitability by modulating neuronal activity under the anode, likely in the cerebellar cortex.
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To our knowledge this is the first demonstration that anodal cerebellar DCS modulates
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excitability of the cervical spinal distribution network. The significance of these findings and
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possible clinical implications are discussed below.
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In agreement with previous similar studies, median nerve stimulation combined with low
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intensity TMS facilitated MEPs evoked in the BB at intensities slightly higher than threshold
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using ISIs designed to summate inputs in PNs [13-15, 17]. An optimal range of ISIs between
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nerve stimulation and TMS was previously reported to lie between 6-9ms [13, 16, 17], based Page 12
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on afferent and efferent conduction times, allowing an additional 3ms of central conduction
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time for summation of inputs at the C3, C4 PNs [5]. The same ISIs were utilised in the
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current study and similar responses were observed, although the suppression was not as
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robust as that in previous studies [13, 16, 17]. The current findings also indicate that
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cerebellar DCS can be employed to modify excitability of cervical PNs in humans, adding to
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knowledge provided by previous studies employing transcranial DCS over M1 [13, 17].
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The finding that anodal cerebellar DCS produced disfacilitation of the PNs was consistent
8
with our a priori hypothesis, based on the known neuroanatomical output projections from
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the cerebellum. Inhibitory Purkinje cells located in the cerebellar cortex modulate activity in
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the deep cerebellar nuclei, which in turn project to the thalamus and M1 (cerebello-thalamo-
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cortical pathway) [29, 30] or brainstem motor nuclei, such as the red nucleus and reticular
12
formation [31, 32]. The rubrospinal tract arises from the red nucleus, crossing over to the
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contralateral ventral tegmental decussation and descends to the spinal cord [31-33]. The
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primary projection is directed to the cervical spine and facilitates motor neurons that
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innervate flexor muscles. The red nucleus itself receives input from the premotor and primary
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motor cortices, as well as cerebellar innervation from the globose and emboliform nuclei [33,
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34]. Furthermore, axons of the cerebellar fastigial nucleus synapse with the reticular
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formation in the brainstem via the fastigial reticular pathway, thereby influencing the
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reticulospinal tract [31]. The reticulospinal tract is known to terminate on PNs as well as
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rostral inhibitory interneurons in the non-human primate [4]. It is reasonable to assume that
21
modulation of cerebellar activity using non-invasive brain stimulation would indirectly
22
influence excitability of the PNs. While the exact mechanisms of how anodal DCS affects
23
cells of the cerebellar cortex is unknown, a parsimonious explanation for our current findings
24
is that anodal stimulation increases Purkinje cell excitability, enhancing inhibition of the deep
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cerebellar nuclei, outputs to cortical and brainstem motor regions and descending projections
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to the spinal cord. Furthermore, the inhibition imposed by Purkinje neurons on the output
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from the deep cerebellar nuclei also modifies motor control through modulation of
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M1excitabilty, whose output directly influences the excitability of PNs [23, 30, 35]. The lack
4
of effect of cerebellar DCS on the inhibitory pathway makes sense from a functional
5
viewpoint. The effect of ‘turning on’ PNs would be to generate synergistic recruitment of
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muscles relevant for a particular functional task. Reduced PN facilitation concurrent with
7
inhibition would serve to ‘switch off’ the same muscle synergy pattern when it is not
8
required. A previous study of bilateral M1 DCS found a similar disfacilitation of PNs without
9
effect on the inhibitory pathway [17]. However, the current result should be interpreted with
10
caution as we did not observe robust inhibition of PNs at baseline. This might indicate the
11
effect of cerebellar DCS on inhibitory interneurons controlling PNs was not probed
12
adequately by the experimental protocol. A future study might induce greater PN suppression
13
by increasing intensity of the peripheral nerve stimulus, rather than increasing TMS intensity,
14
[15] to further confirm that cerebellar DCS does not influence inhibition of PNs. Our current
15
result does indicate that the cerebellum modulated PN activity and may assist M1 to control
16
ipsilateral upper limb muscle synergies in healthy adults.
17
There was no effect of cerebellar DCS on performance of the pursuit rotor task. This was
18
unexpected as we hypothesised the coordination task would be degraded secondary to
19
reduced drive to PNs from higher centres. However, previous studies have reported
20
improvements in upper limb motor learning tasks in healthy adults [36] and subtle
21
improvements in handwriting in people with writing dystonia [37] following cerebellar DCS.
22
A reasonable explanation might be that anodal cerebellar DCS improved explicit learning of
23
the task and this offset any decrement in physical performance due to PN disfacilitation. This
24
question can be addressed by using a task that involves more overt co-ordination between
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proximal and distal muscles, that also rely on cerebellar functions such as error learning
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during reaching movements [36, 38, 39]. Alternatively, skilled upper limb tasks that do not
2
involve repetition, hence avoiding a learning effect might better expose functional
3
consequences of altered PN excitability following cerebellar modulation. Further studies
4
should be undertaken to assess effects of cerebellar anodal DCS on upper limb coordination
5
tasks, as findings may have relevance for using cerebellar stimulation as a therapeutic
6
intervention in movement disorders characterised by abnormal synergies such as after stroke
7
and in focal arm dystonia.
8
A limitation of this study is that we did not include a direct measure of cerebello-thalamo-
9
cortical excitability, for example double coil, paired-pulse TMS [29, 30]. Therefore, we
10
cannot be certain that anodal DCS modified activity in the cerebellar cortex. Even though we
11
believed we were stimulating the cerebellum with the specific electrode placements, without
12
structural scans, electrophysiology or any other objective measure, it is not possible to be
13
absolutely confident. Our results could also be explained by direct stimulation of the RF and
14
brainstem. However, we believe this was unlikely as our previous studies have demonstrated
15
that cerebellar-brain inhibition evoked using paired pulse TMS, was reduced by anodal
16
cerebellar DCS [22, 37]. As the same method of anodal cerebellar DCS was employed in the
17
current study, it is likely that cerebellar excitability was modified in similar ways.
18
Furthermore, computer modelling studies have also indicated that the DCS configuration
19
employed within out study directly influences the cerebellar cortex [40], providing indirect
20
support for cerebellar stimulation in the current study. A second limitation is that we tested
21
the non-dominant hemisphere as we aimed to examine the effect of cerebellar DCS on
22
coordination with the non-dominant hand as a surrogate model for an impaired limb.
23
However, PN modulation is greater in the dominant compared to non-dominant hemisphere
24
[41], and this might explain why the suppression was not as robust in the current study as for
25
our previous investigations [13, 16, 17]. A third limitation pertains to the electrode position
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1
for the control condition, which was 3cm lateral from the mid-sagittal plane. This position
2
was chosen as to completely avoid cerebellar stimulation and assumed to stimulate the
3
parietal lobe instead. However, this electrode placement may have partially overlapped with
4
Brodmann’s Area 4, corresponding with M1 [42], which may explain the mild decrease in PN
5
inhibition. However, if anodal DCS at the control site had affected ipsilateral M1, then
6
activity in the inhibitory pathway should increase, not decrease, given previous findings that
7
cathodal DCS over ipsilateral M1 decreases this activity [13].
8
Conclusion
9
The results of this study confirm the cerebellum indirectly modulates propriospinal networks
10
in humans, an important finding for the understanding of human upper limb coordination. It
11
is well known that the cerebellum has a pivotal role in implementing sensorimotor strategies
12
by constantly monitoring sensory feedback to update and correct motor output for skilled
13
execution of upper limb tasks and inter-limb coordination. Further studies to replicate the
14
findings using different motor tasks are required to improve understanding of how indirect
15
modulation of propriospinal networks by the cerebellum influences formation of upper limb
16
muscle synergies. This knowledge may support the use of anodal cerebellar DCS as a novel
17
treatment intervention for movement disorders exhibiting abnormal muscle synergy
18
formation such as stroke and dystonia.
19
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1
Acknowledgements
2 3
The authors thank Professor Winston Byblow and Associate Professor Cathy Stinear of the University of Auckland for providing the rotor task application for this study.
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
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[18] Alstermark B, Isa T, Tantisira B. Projection from excitatory C3-C4 propriospinal neurones to spinocerebellar and spinoreticular neurones in the C6-Th1 segments of the cat. Neurosci Res. 1990;8:124-30. [19] Alstermark B, Lindström S, Lundberg A, Sybirska E. Integration in descending motor pathways controlling the forelimb in the cat. 8. Ascending projection to the lateral reticular nucleus from C3-C4 propriospinal also projecting to forelimb motoneurones. Experimental brain research. 1981;42:282. [20] Alstermark B, Ekerot C. Organization of the ascending projection from C3–C4 propriospinal neurones to cerebellum via the lateral reticular nucleus. Acta Physiol Scand. 1992;146:P2. [21] Ekerot C-F. The lateral reticular nucleus in the cat. Experimental brain research. 1990;79:109-19. [22] Bradnam L, Young J, Doeltgen S. Anodal Direct Current Stimulation of the Cerebellum reduces Cerebellar-Brain Inhibition but does not influence afferent input from the hand or face in health adults. Cerebellum. 2015;In press. [23] Galea JM, Jayaram G, Ajagbe L, Celnik P. Modulation of cerebellar excitability by polarity-specific noninvasive direct current stimulation. The Journal of Neuroscience. 2009;29:9115-22. [24] Raz N, Williamson A, Gunning-Dixon F, Head D, Acker JD. Neuroanatomical and cognitive correlates of adult age differences in acquisition of a perceptual-motor skill. Microscopy research and technique. 2000;51:85-93. [25] Nicholls ME, Thomas NA, Loetscher T, Grimshaw GM. The Flinders Handedness survey (FLANDERS): a brief measure of skilled hand preference. Cortex. 2013;49:2914-26. [26] Rossini PM, Barker A, Berardelli A, Caramia M, Caruso G, Cracco R, et al. Non-invasive electrical and magnetic stimulation of the brain, spinal cord and roots: basic principles and procedures for routine clinical application. Report of an IFCN committee. Electroencephalography and clinical neurophysiology. 1994;91:79-92. [27] Gandiga PC, Hummel FC, Cohen LG. Transcranial DC stimulation (tDCS): a tool for double-blind sham-controlled clinical studies in brain stimulation. Clinical Neurophysiology. 2006;117:845-50. [28] Rom DM. A sequentially rejective test procedure based on a modified Bonferroni inequality. Biometrika. 1990;77:663-5. [29] Ugawa Y, Uesaka Y, Terao Y, Hanajima R, Kanazawa I. Magnetic stimulation over the cerebellum in humans. Annals of neurology. 1995;37:703-13. [30] Daskalakis ZJ, Paradiso GO, Christensen BK, Fitzgerald PB, Gunraj C, Chen R. Exploring the connectivity between the cerebellum and motor cortex in humans. The Journal of physiology. 2004;557:689-700. [31] Snell RS. Clinical neuroanatomy: Lippincott Williams & Wilkins; 2010. [32] Siegel A, Sapru HN. Essential neuroscience: Lippincott Williams & Wilkins; 2006. [33] Keifer J, Houk JC. Motor function of the cerebellorubrospinal system. Physiological reviews. 1994;74:509-42. [34] Benarroch EE. Basic neurosciences with clinical applications: Elsevier Health Sciences; 2006. [35] Oulad Ben Taib N, Manto M. Trains of epidural DC stimulation of the cerebellum tune corticomotor excitability. Neural plasticity. 2013;2013. [36] Galea JM, Vazquez A, Pasricha N, de Xivry J-JO, Celnik P. Dissociating the roles of the cerebellum and motor cortex during adaptive learning: the motor cortex retains what the cerebellum learns. Cerebral Cortex. 2011;21:1761-70. [37] Bradnam LV, Graetz LJ, McDonnell MN, Ridding MC. Anodal transcranial direct current stimulation to the cerebellum improves handwriting and cyclic drawing kinematics in focal hand dystonia. Frontiers in human neuroscience. 2015;9:286. [38] Schlerf JE, Galea JM, Bastian AJ, Celnik PA. Dynamic modulation of cerebellar excitability for abrupt, but not gradual, visuomotor adaptation. The Journal of Neuroscience. 2012;32:11610-7. [39] Sadnicka A, Patani B, Saifee TA, Kassavetis P, Parees I, Korlipara P, et al. Normal motor adaptation in cervical dystonia: a fundamental cerebellar computation is intact. The Cerebellum. 2014;13:558-67.
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[40] Parazzini M, Rossi E, Ferrucci R, Liorni I, Priori A, Ravazzani P. Modelling the electric field and the current density generated by cerebellar transcranial DC stimulation in humans. Clinical Neurophysiology. 2014;125:577-84. [41] Marchand-Pauvert V, Mazevet D, Pierrot-Deseilligny E, Pol S, Pradat-Diehl P. Handednessrelated asymmetry in transmission in a system of human cervical premotoneurones. Experimental brain research. 1999;125:323-34. [42] DaSilva AF, Volz MS, Bikson M, Fregni F. Electrode positioning and montage in transcranial direct current stimulation. Journal of visualized experiments: JoVE. 2011.
9 10
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1
Figure 1. Schematic diagram showing cerebellar control of putative PNs. The PNs and
2
inhibitory interneurons project to the lateral reticular nucleus, which in turn projects to the
3
cerebellum. Outputs of the cerebellar cortex, via Purkinje neurons provide tonic inhibition of
4
the deep cerebellar nuclei. There are ascending commands to M1 via the dentatothalamic
5
tract and descending commands to PNs indirectly via the corticospinal tract, and brainstem
6
reticulospinal and rubrospinal tracts. These descending pathways modulate excitability of
7
PNs and associated inhibitory interneurons. Not all inhibitory connections are shown for
8
clarity. V shape connections signify excitation, circles signify inhibition. M1, primary motor
9
cortex. T, thalamus. DCN, Deep cerebellar nuclei. RN, red nucleus. RF, reticular formation.
10
LRN, lateral reticular nucleus. PN, propriospinal neuron. αMNs, alpha motoneurons. DTT,
11
dentatothalamic tract. PkN, Purkinje neuron. CST, cerebrospinal tract. RbST, rubrospinal
12
tract. RST, reticulospinal tract. InhN, inhibitory neuron.
13
Figure 2. Average rectified EMG traces of left BB MEPs from 1 representative subject
14
showing FAC and INH, at both pre-DCS (top trace) and post-DCS (bottom trace). NC (grey)
15
and C (black) are shown for FAC and INH at each time point. The MEPAREA was calculated
16
within the time window depicted between the dashed vertical lines for this subject. The ratio
17
of the conditioned to non-conditioned MEPAREA (C/NC) is provided at the right of each trace.
18
Figure 3. A. Pre and post-DCS FAC (black) and INH (grey) MEPs across sham, real and
19
control. Pre-DCS, FAC and INH differed from baseline in all sessions (P < 0.01) apart from
20
sham INH (P = 0.30). Real FAC differed significantly from pre to post-DCS (*P = 0.001).
21
Control INH differed pre to post-DCS (**P = 0.015). Each bar represents the group average
22
(n=12). Error bars indicate SE. B. ΔFAC (black) and ΔINH (grey) across sham, real and
23
control. Positive values indicate an increase in FAC or INH, negative values indicate a
24
decrease. There was a difference in ΔFAC between real and sham (*P = 0.001) as well as real
25
and control sessions (**P = 0.009). Each bar represents the group average (n=12). Error bars Page 20
Page 20 of 27
1
indicate SE. C. ΔSlope across SHAM, real and control. Positive values indicate an increase in
2
Slope, negative values indicate a decrease. Slope did not change significantly between real
3
and sham (P = 0.18) or real and control (P = 0.07). Each bar represents the group average
4
(n=12). Error bars indicate SE.
5
Table 1. Ratios for FAC and INH for the three conditions, pre and post cerebellar DCS and
6
the difference expressed as post – pre. One sample t-tests were used to determine the
7
presence of FAC and INH at baseline. Post hoc paired-sample t-tests were used to assess
8
effects of stimulation on each element. Significance at < 0.05 is signified by * and < 0.001 is
9
signified by †. There was no difference between conditions at baseline for either FAC or INH
10
(all P > 0.11).
11
Table 2. Non-conditioned (NC) MEP (V.ms) and rmsEMG (mV) means ± SE across sham,
12
real and control conditions.
13
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1
Figure 1
2 3
Page 22
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1
Figure 2 C NC
0.5mV 10ms
Pre-tDCS FAC
1.66
Pre-tDCS INH
0.70
Post-tDCS FAC
1.14
Post-tDCS INH
0.76
2
3
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1
2 3
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1
Figure 3 **
*
A 1.6
B
1.4
0.1
1.2 *
1
0
∆ C/NC
C/NC
**
*
0.2
0.8 0.6
Sham
Real
Control
-0.1
0.4
-0.2
0.2
-0.3
0 Pre Sham
2
Post
Pre Real FAC
Post
Pre Control
-0.4
Post
FAC
INH
INH
C 0.2 0.1
∆ Slope
0 -0.1
Sham
Real
Control
-0.2 -0.3 -0.4
3 4 5
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1
Table 1 Pre FAC
Post INH
FAC
INH
Difference (Post – Pre) FAC INH
Real
1.40†
0.93*
1.13
0.99
-0.28†
0.07
Control
1.29†
0.94†
1.34
1.05
0.04
0.11
Sham
1.38†
0.97
1.35
1.04
-0.03
0.07
2 3 4
Table 2 NC MEP Pre
NC MEP Post
rmsEMG Pre
rmsEMG Post
SHAM
2.45 ± 0.38
2.77 ± 0.39
0.097 ± 0.036
0.10 ± 0.041
Real
2.35 ± 0.33
2.55 ± 0.19
0.088 ± 0.038
0.091 ± 0.026
Cond
2.68 ± 0.48
2.41 ± 0.33
0.093 ± 0.037
0.094 ± 0.036
SHAM
4.30 ± 0.50
4.73 ± 0.64
0.094 ± 0.033
0.94 ± 0.038
Real
4.14 ± 0.34
4.81 ± 0.41
0.087 ± 0.027
0.095 ± 0.025
Cond
4.88 ± 0.93
4.19 ± 0.94
0.096 ± 0.039
0.092 ± 0.036
FAC
INH
5
Page 26
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1
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S1935-861X(16)00006-1 http://dx.doi.org/doi: 10.1016/j.brs.2016.01.002 BRS 844
To appear in:
Brain Stimulation
Received date: Revised date: Accepted date:
20-10-2015 1-1-2016 4-1-2016
Please cite this article as: Muhammed Chothia, Sebastian Doeltgen, Lynley V Bradnam, Anodal Cerebellar Direct Current Stimulation Reduces Facilitation of Propriospinal Neurons in Healthy Humans, Brain Stimulation (2016), http://dx.doi.org/doi: 10.1016/j.brs.2016.01.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Title: Anodal cerebellar direct current stimulation reduces facilitation of propriospinal
2
neurons in healthy humans
3 4
Authors and affiliations
5
Muhammed Chothia1, Sebastian Doeltgen2 Lynley V Bradnam1,3
6
1
7
Flinders University, Adelaide, South Australia, 5041
8
2
9
University, South Australia, 5041
Applied Brain Research Laboratory, Discipline of Physiotherapy, Faculty of Health Sciences
Discipline of Speech Pathology and Audiology, School of Health Sciences, Flinders
10
3
11
NSW, Australia
Discipline of Physiotherapy, Graduate School of Health, University of Technology Sydney,
12 13
Corresponding Author
14
Lynley Bradnam, Discipline of Physiotherapy, Graduate School of Health, University of
15
Technology Sydney, PO Box 123, NSW, Australia, 2007.
16
Email: [email protected]
17 18
Running Title: Cerebellar stimulation and propriospinal neurons
19 20 21 22 23 24
Page 1
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1
Highlights
2 3
4 5
6 7
8 9
Activity in cervical propriospinal neurons (PNs) was explored using paired TMS and peripheral nerve stimulation Anodal cerebellar DCS was used to probe cerebellar control of propriospinal activity Facilitation of PNs was reduced by cerebellar anodal DCS, with no effect on inhibition The cerebellum may contribute to upper limb coordination by modulation of PN excitability in humans
10 11
Abstract
12
Background
13
Coordinated muscle synergies in the human upper limb are controlled, in part, by a neural
14
distribution network located in the cervical spinal cord, known as the cervical propriospinal
15
system. Studies in cats and non-human primates indicate the cerebellum is indirectly
16
connected to this system via output pathways to the brainstem. Therefore, the cerebellum
17
may indirectly modulate excitability of putative propriospinal neurons (PNs) in humans
18
during upper limb coordination tasks.
19
Objective/Hypothesis
20
This study aimed to test whether anodal direct current stimulation (DCS) of the cerebellum
21
modulates PNs and upper limb coordination in healthy adults. The hypothesis was that
22
cerebellar anodal DCS would reduce descending facilitation of PNs and improve upper limb
23
coordination.
24
Methods
25
Transcranial magnetic stimulation (TMS), paired with peripheral nerve stimulation probed
26
activity in facilitatory and inhibitory descending projections to PNs following an established
Page 2
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1
protocol. Coordination was tested using a pursuit rotor task performed by the non-dominant
2
(ipsilateral) hand. Anodal and sham DCS were delivered over the cerebellum ipsilateral to the
3
non-dominant hand in separate experimental sessions. Anodal DCS was applied to a control
4
site lateral to the vertex in a third session. Twelve right-handed healthy adults participated.
5
Results
6
Pairing TMS with sub-threshold peripheral nerve stimulation facilitated motor evoked
7
potentials at intensities just above threshold in accordance with the protocol. Anodal
8
cerebellar DCS reduced facilitation without influencing inhibition, but the reduction in
9
facilitation was not associated with performance of the pursuit rotor task.
10
Conclusions
11
The results of this study indicate dissociated indirect control over cervical PNs by the
12
cerebellum in humans. Anodal DCS of the cerebellum reduced excitability in the facilitatory
13
descending pathway with no effect on the inhibitory pathway to cervical PNs. The reduction
14
in PN excitability is likely secondary to modulation of primary motor cortex or brainstem
15
nuclei, and identifies a neuroanatomical pathway for the cerebellum to assist in coordination
16
of upper limb muscle synergies in humans.
17 18
Keywords: propriospinal; direct current stimulation; transcranial magnetic stimulation;
19
cerebellum; stroke; dystonia
20 21
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1
Introduction
2
Reaching and grasping to manipulate objects with the hand is a fundamental task in humans,
3
involving fine-tuned stereotypical movements demanding precise co-contraction of proximal
4
and distal musculature. Coordinated movement relies on synergistic activation of each muscle
5
involved in a functional task, as well as the coordinated inhibition of their respective
6
antagonists. Abnormal patterns of movement exist between proximal and distal upper limb
7
muscles in neurological disorders such as chronic stroke and dystonia [1, 2], limiting useful
8
function [3]. One theory for upper extremity muscle synergies are that they are formed by
9
pre-motoneuronal spinal distribution networks in the upper cervical cord [4, 5], known as the
10
cervical propriospinal system. Propriospinal neurons (PNs) were described in cats [6] and
11
non-human primates [7, 8] and found to contribute to muscle synergies for upper limb target
12
reaching by exciting α-motor neurons (αMNs) innervating agonist muscles and suppressing
13
antagonist muscle activity [9]. Propriospinal neurons integrate descending cortical commands
14
with afferent feedback from the upper limb, to permit immediate update of the motor
15
command [6, 10, 11] (Figure 1). Both descending (efferent) and ascending (afferent) inputs
16
can directly excite PNs or suppress their activity via local inhibitory interneurons [8, 10, 12]
17
so that synergies are modulated according to task requirements.
18
Activity in cervical PNs can be probed indirectly in humans using transcranial magnetic
19
stimulation (TMS), paired with peripheral nerve stimulation. With an appropriate
20
interstimulus interval (ISI), the two stimuli summate in the putative PNs [13-17]. The ISI
21
optimal range is calculated to be 6-9ms, based on the afferent and efferent conduction times
22
of peripheral nerves and spinal cord white matter [5]. Previous studies demonstrated TMS at
23
intensities just above threshold, paired with sub-motor threshold nerve stimulation, facilitates
24
motor evoked potentials (MEPs). Slightly stronger TMS intensities paired with the same sub-
25
motor threshold nerve stimulation suppress MEPs, presumably by recruitment of local Page 4
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1
inhibitory interneurons [15]. In humans, PNs are held under tonic inhibition which is released
2
during functional tasks that require synergistic activation of proximal and distal upper limb
3
muscles [14, 16].
4
Experiments in cats and non-human primates indicate the cerebellum is intimately involved
5
in the spinal distribution network. Propriospinal neurons send ascending projections to the
6
lateral reticular nucleus in the brainstem which projects in turn to the cerebellum [18, 19].
7
The extensive major mossy fibre connections between the lateral reticular nucleus and the
8
cerebellum permits an efferent copy of the motor command from PNs to spinal αMNs to be
9
analysed by the cerebellum [20, 21]. The cerebellum can update the motor command by its
10
output projections to the primary motor cortex (M1) and brainstem motor nuclei and their
11
descending pathways which terminate on PNs and αMNs [8] (Figure 1). However, direct
12
evidence of modulation of PN activity by the cerebellum has not yet been demonstrated in
13
humans. This question may be answered with non-invasive stimulation. The cerebellum can
14
be modulated by anodal direct current stimulation (DCS) in healthy humans [22, 23] but there
15
are no studies that have investigated the influence of cerebellar DCS on putative PNs in
16
humans.
17
The objective of this study was to examine the role of the cerebellum by probing the effect of
18
anodal cerebellar DCS on facilitatory and inhibitory projections to PNs in healthy humans.
19
Cerebellar DSC influence on ipsilateral upper limb coordination was tested using a pursuit
20
rotor task performed with the non-dominant hand [24]. The pursuit rotor task was chosen as it
21
requires precise coordination of hand and arm, considered to rely, in part, on cerebellar
22
function. The a priori hypothesis was that anodal cerebellar DCS would reduce PN
23
excitability, because outputs from the cerebellar cortex are inhibitory and would reduce
24
descending drive from M1 or brainstem nuclei to the spinal cord. We also hypothesised that
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1
reduced facilitation of PNs would degrade performance of the non-dominant hand on the
2
pursuit rotor task, due to impaired muscle synergies and coordination.
3
Methods
4
Participants
5
Twelve healthy adults (mean age = 29.6 yr, range = 23-57 yr, 7 males) without history of
6
neurological or musculoskeletal disorder affecting the neck or upper limb participated in the
7
study, following screening for contraindications to TMS by a physician. All twelve
8
participants were right handed, assessed with the Flinders Handedness survey (9.83 ± 0.11)
9
[25]. Informed, written consent was obtained from each individual. Ethical approval was
10
provided by the Southern Adelaide Clinical Human Research Ethics Committee. The study
11
was conducted in accordance with the Declaration of Helsinki.
12
Experimental Design
13
Participants completed three experimental sessions separated by at least 5 days.
14
Neurophysiological and behavioural data were collected pre- and post-DCS at each session.
15
Participants were randomly assigned to intervention order and blinded to the stimulation at
16
each session. The same experimenter delivered the intervention and collected outcome
17
measures and was not blinded.
18
Electromyography
19
Surface electromyography (EMG) was recorded from the left biceps brachii (BB) and left
20
flexor carpi radialis (FCR) using disposable electrodes (30 x 20mm; Ambu, Ballerup,
21
Denmark). EMG recordings were made using a belly-tendon montage. EMG from the FCR
22
was only used to set the intensity for median nerve stimulation; no data from this muscle was
23
collected or analysed. EMG signals were amplified (CED 1902; Cambridge Electronic
Page 6
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1
Design (CED), Cambridge, United Kingdom), band-pass-filtered (20 –1,000 Hz) and sampled
2
at 2 kHz (CED 1401).
3
Transcranial Magnetic Stimulation
4
Single-pulse TMS was delivered to the right M1 to elicit MEPs in the left BB with a 70mm
5
figure-of-eight remote control coil and a MagStim 2002 unit (MagStim, Dyfed, Wales). The
6
coil was held over the scalp at a 45o angle to induce a posterior to anterior current in the
7
underlying motor cortex. Participants held a 1000g weight in their left hand with the forearm
8
in full supination, the wrist in slight flexion and the elbow flexed between 90 and 120o, in
9
order to co-contract the BB and FCR during TMS [13, 17]. The optimal stimulation site
10
evoking the largest MEP in the left BB was located and marked on the scalp. Active motor
11
threshold (AMT) was defined for the left BB as the minimum stimulus intensity that elicited
12
a 100µV response, with a size, shape and latency that was consistent with an MEP, in 5 out of
13
10 trials [26], while maintaining the task position. Once AMT was determined, six stimulus
14
intensities were calculated in increments of 2% maximal stimulator output (MSO), starting
15
from AMT -2% of MSO. Each TMS intensity was delivered in random order as a separate
16
block during task performance. The blocks consisted of 16 non-conditioned MEPs (NC) and
17
16 MEPs conditioned (C) by median nerve stimulation (MNS), recorded at a rate of 0.2Hz.
18
Rest breaks were provided between blocks to prevent fatigue. Responses were recorded using
19
Signal Software v6 (CED) and saved for offline analysis.
20
Median Nerve Stimulation
21
The median nerve was stimulated using square-wave pulses (1ms) delivered by a Digitimer
22
DS7A constant current stimulator (Digitimer, Hertfordshire, UK) prior to TMS. Timing
23
between the two stimuli was automated using Signal software (V6). A motor point pen
24
electrode (Compex) was used to locate the median nerve in the cubital fossa at the elbow.
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1
The optimum site for MNS was detected by observation of the motor response (M-wave) on
2
the computer screen. The cathode was fixed to the skin by an adhesive electrode (Ambu,
3
Ballerup, Denmark) over the optimal stimulation point for the median nerve at the elbow. The
4
cathode was located proximal, and the anode distal, in relation to the spinal cord. Motor
5
threshold was defined as the minimum current intensity to induce an M-wave response while
6
maintaining the task position. The intensity for MNS was set to 0.8 of motor threshold, to
7
preferentially stimulate group I sensory afferents [15]. To determine the optimum ISI
8
between MNS and TMS for each individual, detailed exploration of different ISIs between 6-
9
9 ms was undertaken using TMS intensities of AMT +2% to +4% MSO in the first
10
experimental session. The ISI that generated maximum facilitation of the non-conditioned
11
MEP was identified as the optimal ISI and used for all three conditions for that individual.
12
The range of 6-9 ms was determined in previous studies [13, 16, 17].
13
Direct Current Stimulation
14
Direct current stimulation was delivered using a TCT stimulator (TCT Research, Hong Kong)
15
with a constant current of 2 mA, for 15 minutes using two rubber electrodes encased in
16
saline-soaked sponges. The anode was 16cm2 and the cathode was 24cm2, producing a
17
current density under the anode of 1.25A/m2. The anode was positioned over the left lateral
18
cerebellum, 3cm lateral to the external occipital protuberance. The cathode was placed over
19
the left buccinator [23]. The sham condition used the identical electrode configuration,
20
ramping the current up to 2mA and back down to 0mA over 30s [27]. To confirm any effect
21
on PNs resulted from cerebellar anodal DCS, an active control condition, where the anode
22
was positioned over the left parietal lobe 3cm lateral from the mid-central scalp (Cz) and the
23
cathode on the left buccinator, was included. Participants sat quietly throughout the
24
intervention and for 5 minutes afterwards.
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1
Rotor pursuit task
2
Participants were seated at a desk in a standardised position with a tablet (iPad Apple Pty
3
Ltd) directly in front of them, laid flat in portrait orientation. Participants were instructed to
4
follow a moving cursor on the tablet screen with a stylus held in their left (non-dominant)
5
hand. The cursor followed a circular path, 6.25cm in radius (Rotor Test, Stroke Science,
6
University of Auckland) at a speed of 21 revolutions per minute (RPM). One practice trial of
7
7 revolutions (21 RPM) was undertaken at the beginning of each experiment. After that, five
8
trials of 7 revolutions (21 RPM) were completed with a 15 second rest between trials, before
9
and after the DCS intervention.
10
Data Analysis
11
Electromyography traces from the left BB were rectified and the MEPAREA calculated between
12
the onset and offset latencies using the same window for each individual, optimised across
13
the three conditions. The MEPAREA was averaged for each block and expressed as a ratio of
14
conditioned to non-conditioned (C/NC) responses. The facilitation element (FAC) was
15
determined by a ratio greater than 1 and the following inhibition element (INH) by a ratio less
16
than 1. The blocks that produced the greatest degree of FAC and INH for each participant
17
were identified and the data averaged for each experimental condition. Traces from a
18
representative subject are provided in Figure 2. The difference in FAC and INH elements pre
19
to post DCS was calculated (Δ = post DCS – pre DCS) for each individual and averaged [17].
20
Root mean square EMG (rmsEMG) was calculated between 100 and 10ms prior to the
21
stimulus artefact for all MEP data.
22
For the pursuit rotor task the time for which the stylus was correctly in contact with the
23
moving cursor (total contact time) was recorded from each of the five individual trials in a
24
trial block. The first trial of each block was used to normalise the four subsequent trials, in
Page 9
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1
order to account for variations in task performance between individuals. The five normalised
2
trials were then plotted against time and subjected to linear regression analysis. The slope of
3
the linear line of best fit was calculated to indicate task performance over the five trials.
4
Group averages were calculated for each condition and time point for statistical analysis. The
5
difference in total contact time over the five trials before and after DCS was calculated as
6
ΔSlope = post-DCS − pre-DCS.
7
Statistical Analysis
8
Ratios, non-conditioned MEPs and rmsEMG were tested for normality using the Shapiro-
9
Wilks test. Where data did not meet assumptions of normality, data was log10 transformed
10
and statistical analysis was performed on the logarithmic transformed data. For pre-
11
intervention ratios, a one sample t-test was used to determine if FAC and INH were present at
12
baseline, and paired sample t-tests compared baseline consistency across the three conditions.
13
MEP ratios and non-conditioned MEPs were analysed using separate 3 CONDITION (Real,
14
Control, Sham), x 2 ELEMENT (FAC, INH) x 2 TIME (pre, post) repeated measures ANOVA
15
(rmANOVA). To compare effects of DCS across conditions, ΔFAC and ΔINH were tested
16
using a 3 CONDITION (Real, Control, Sham) x 2 ELEMENT (ΔFAC, ΔINH) rmANOVA.
17
Finally, root mean square EMG (rmsEMG) was analysed using a separate 3 CONDITION (Real,
18
Control, Sham) x 2 MEP (Cond, NC) x 2 TIME (pre, post) rmANOVA for each ELEMENT. The
19
difference in slope value for the pursuit rotor task pre to post DCS was first analysed using
20
the non-parametric Wilcoxon signed-rank test, separately for each condition. Second, ΔSlope
21
was analysed using a 3 CONDITION (Real, Control, Sham) rmANOVA. Spearman’s
22
correlation was used to test for relationships between TMS parameters demonstrating
23
significant change and task performance. The Greenhouse Geisser correction was utilised
24
where data did not meet conditions of sphericity. Post hoc paired t-tests were used to explore
Page 10
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1
significant main effects or interactions. Multiple tests were adjusted for significance using a
2
modified Bonferroni test [28]. Data are expressed as mean ± SE.
3
Results
4
One sample t-tests revealed FAC and INH at baseline for all conditions (all P < 0.01), apart
5
from the INH element in the Sham condition (P = 0.22). Paired sample t-tests revealed no
6
difference for CONDITION or ELEMENT pre-stimulation (all P > 0.11) (Table 1). The
7
rmANOVA revealed a main effect of ELEMENT (F1,11 = 116.56, P < 0.0001) and CONDITION
8
(F2,22 = 3.46, P = 0.049), a CONDITION by TIME interaction (F2,22 = 5.88, P = 0.014) and a TIME
9
by ELEMENT interaction (F1,11 = 21.10, P = 0.001). There was a strong trend for a CONDITION
10
by ELEMENT by TIME interaction (P = 0.05). There was no main effect for TIME (P = 0.82) or
11
an interaction between CONDITION and ELEMENT (P = 0.54) (Figure 3A). To assess the
12
intervention effect, post hoc, paired sample t-tests revealed a reduction across TIME for the
13
FAC ELEMENT following anodal cerebellar DCS (P < 0.001) but not for Sham and control
14
DCS (both P > 0.33) (Figure 3B). For the INH ELEMENT, there was a reduction following
15
control DCS (P = 0.015) but not for anodal cerebellar DCS (P = 0.1) or sham DCS (P = 0.52)
16
(Table 1).
17
The rmANOVA for ΔFAC and ΔINH revealed a main effect of ELEMENT (F1,11 = 21.78, P =
18
0.001) and CONDITION (F2,22 = 5.86, P = 0.014), but no interaction (P = 0.07). The reduction
19
in facilitation represented by ΔFAC after anodal cerebellar DCS differed from sham (P =
20
0.001) and control (P = 0.009) conditions (Figure 3C). There was no difference for ∆INH
21
between the three conditions (all P > 0.19). For the NC MEPs, there was a main effect of
22
ELEMENT
23
to evoke INH were larger (4.51 +/- 0.47 V.ms) than NC MEPs at the lower TMS intensity to
24
evoke FAC (2.54 +/- 0. 34 V.ms). There were no other main effects or any interactions (all P
(F1,11 = 95.33, P < 0.0001). As expected, NC MEPs at higher TMS intensities used
Page 11
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1
> 0.07) (Table 2). Data for the prestimulus rmsEMG were log10 transformed prior to analysis
2
and the rmANOVA found prestimulus rmsEMG was consistent across condition and time for
3
both FAC and INH components (both P > 0.11) (Table 2). For the pursuit rotor task, the
4
Wilcoxon signed-rank tests revealed no change in slope after stimulation for all conditions
5
(all Z < -0.94, all P > 0.1). For ΔSlope, there was no difference between conditions (P = 0.07)
6
although observation of the data indicates slope was increased following anodal cerebellar
7
DCS and decreased following sham cerebellar DCS (Figure 3D). A Spearman's rank-order
8
correlation revealed there was no relationship between ΔFAC and ΔSlope (rs = 0.469, P =
9
0.124) in the anodal cerebellar DCS condition.
10
Discussion
11
We investigated if anodal cerebellar DCS influenced descending facilitatory and inhibitory
12
drive to cervical PNs in healthy humans and whether this was related to performance of an
13
upper limb coordination task. The major new finding was that anodal cerebellar DCS reduced
14
activity (or disfacilitation) in excitatory descending pathways to PNs, without influencing
15
inhibitory pathways. Neither sham cerebellar DCS, nor an active control condition where the
16
anode was placed lateral to the midline at the vertex, altered activity in either descending
17
pathway. These findings suggest that anodal cerebellar DCS indirectly influenced PN
18
excitability by modulating neuronal activity under the anode, likely in the cerebellar cortex.
19
To our knowledge this is the first demonstration that anodal cerebellar DCS modulates
20
excitability of the cervical spinal distribution network. The significance of these findings and
21
possible clinical implications are discussed below.
22
In agreement with previous similar studies, median nerve stimulation combined with low
23
intensity TMS facilitated MEPs evoked in the BB at intensities slightly higher than threshold
24
using ISIs designed to summate inputs in PNs [13-15, 17]. An optimal range of ISIs between
25
nerve stimulation and TMS was previously reported to lie between 6-9ms [13, 16, 17], based Page 12
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1
on afferent and efferent conduction times, allowing an additional 3ms of central conduction
2
time for summation of inputs at the C3, C4 PNs [5]. The same ISIs were utilised in the
3
current study and similar responses were observed, although the suppression was not as
4
robust as that in previous studies [13, 16, 17]. The current findings also indicate that
5
cerebellar DCS can be employed to modify excitability of cervical PNs in humans, adding to
6
knowledge provided by previous studies employing transcranial DCS over M1 [13, 17].
7
The finding that anodal cerebellar DCS produced disfacilitation of the PNs was consistent
8
with our a priori hypothesis, based on the known neuroanatomical output projections from
9
the cerebellum. Inhibitory Purkinje cells located in the cerebellar cortex modulate activity in
10
the deep cerebellar nuclei, which in turn project to the thalamus and M1 (cerebello-thalamo-
11
cortical pathway) [29, 30] or brainstem motor nuclei, such as the red nucleus and reticular
12
formation [31, 32]. The rubrospinal tract arises from the red nucleus, crossing over to the
13
contralateral ventral tegmental decussation and descends to the spinal cord [31-33]. The
14
primary projection is directed to the cervical spine and facilitates motor neurons that
15
innervate flexor muscles. The red nucleus itself receives input from the premotor and primary
16
motor cortices, as well as cerebellar innervation from the globose and emboliform nuclei [33,
17
34]. Furthermore, axons of the cerebellar fastigial nucleus synapse with the reticular
18
formation in the brainstem via the fastigial reticular pathway, thereby influencing the
19
reticulospinal tract [31]. The reticulospinal tract is known to terminate on PNs as well as
20
rostral inhibitory interneurons in the non-human primate [4]. It is reasonable to assume that
21
modulation of cerebellar activity using non-invasive brain stimulation would indirectly
22
influence excitability of the PNs. While the exact mechanisms of how anodal DCS affects
23
cells of the cerebellar cortex is unknown, a parsimonious explanation for our current findings
24
is that anodal stimulation increases Purkinje cell excitability, enhancing inhibition of the deep
25
cerebellar nuclei, outputs to cortical and brainstem motor regions and descending projections
Page 13
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1
to the spinal cord. Furthermore, the inhibition imposed by Purkinje neurons on the output
2
from the deep cerebellar nuclei also modifies motor control through modulation of
3
M1excitabilty, whose output directly influences the excitability of PNs [23, 30, 35]. The lack
4
of effect of cerebellar DCS on the inhibitory pathway makes sense from a functional
5
viewpoint. The effect of ‘turning on’ PNs would be to generate synergistic recruitment of
6
muscles relevant for a particular functional task. Reduced PN facilitation concurrent with
7
inhibition would serve to ‘switch off’ the same muscle synergy pattern when it is not
8
required. A previous study of bilateral M1 DCS found a similar disfacilitation of PNs without
9
effect on the inhibitory pathway [17]. However, the current result should be interpreted with
10
caution as we did not observe robust inhibition of PNs at baseline. This might indicate the
11
effect of cerebellar DCS on inhibitory interneurons controlling PNs was not probed
12
adequately by the experimental protocol. A future study might induce greater PN suppression
13
by increasing intensity of the peripheral nerve stimulus, rather than increasing TMS intensity,
14
[15] to further confirm that cerebellar DCS does not influence inhibition of PNs. Our current
15
result does indicate that the cerebellum modulated PN activity and may assist M1 to control
16
ipsilateral upper limb muscle synergies in healthy adults.
17
There was no effect of cerebellar DCS on performance of the pursuit rotor task. This was
18
unexpected as we hypothesised the coordination task would be degraded secondary to
19
reduced drive to PNs from higher centres. However, previous studies have reported
20
improvements in upper limb motor learning tasks in healthy adults [36] and subtle
21
improvements in handwriting in people with writing dystonia [37] following cerebellar DCS.
22
A reasonable explanation might be that anodal cerebellar DCS improved explicit learning of
23
the task and this offset any decrement in physical performance due to PN disfacilitation. This
24
question can be addressed by using a task that involves more overt co-ordination between
25
proximal and distal muscles, that also rely on cerebellar functions such as error learning
Page 14
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1
during reaching movements [36, 38, 39]. Alternatively, skilled upper limb tasks that do not
2
involve repetition, hence avoiding a learning effect might better expose functional
3
consequences of altered PN excitability following cerebellar modulation. Further studies
4
should be undertaken to assess effects of cerebellar anodal DCS on upper limb coordination
5
tasks, as findings may have relevance for using cerebellar stimulation as a therapeutic
6
intervention in movement disorders characterised by abnormal synergies such as after stroke
7
and in focal arm dystonia.
8
A limitation of this study is that we did not include a direct measure of cerebello-thalamo-
9
cortical excitability, for example double coil, paired-pulse TMS [29, 30]. Therefore, we
10
cannot be certain that anodal DCS modified activity in the cerebellar cortex. Even though we
11
believed we were stimulating the cerebellum with the specific electrode placements, without
12
structural scans, electrophysiology or any other objective measure, it is not possible to be
13
absolutely confident. Our results could also be explained by direct stimulation of the RF and
14
brainstem. However, we believe this was unlikely as our previous studies have demonstrated
15
that cerebellar-brain inhibition evoked using paired pulse TMS, was reduced by anodal
16
cerebellar DCS [22, 37]. As the same method of anodal cerebellar DCS was employed in the
17
current study, it is likely that cerebellar excitability was modified in similar ways.
18
Furthermore, computer modelling studies have also indicated that the DCS configuration
19
employed within out study directly influences the cerebellar cortex [40], providing indirect
20
support for cerebellar stimulation in the current study. A second limitation is that we tested
21
the non-dominant hemisphere as we aimed to examine the effect of cerebellar DCS on
22
coordination with the non-dominant hand as a surrogate model for an impaired limb.
23
However, PN modulation is greater in the dominant compared to non-dominant hemisphere
24
[41], and this might explain why the suppression was not as robust in the current study as for
25
our previous investigations [13, 16, 17]. A third limitation pertains to the electrode position
Page 15
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1
for the control condition, which was 3cm lateral from the mid-sagittal plane. This position
2
was chosen as to completely avoid cerebellar stimulation and assumed to stimulate the
3
parietal lobe instead. However, this electrode placement may have partially overlapped with
4
Brodmann’s Area 4, corresponding with M1 [42], which may explain the mild decrease in PN
5
inhibition. However, if anodal DCS at the control site had affected ipsilateral M1, then
6
activity in the inhibitory pathway should increase, not decrease, given previous findings that
7
cathodal DCS over ipsilateral M1 decreases this activity [13].
8
Conclusion
9
The results of this study confirm the cerebellum indirectly modulates propriospinal networks
10
in humans, an important finding for the understanding of human upper limb coordination. It
11
is well known that the cerebellum has a pivotal role in implementing sensorimotor strategies
12
by constantly monitoring sensory feedback to update and correct motor output for skilled
13
execution of upper limb tasks and inter-limb coordination. Further studies to replicate the
14
findings using different motor tasks are required to improve understanding of how indirect
15
modulation of propriospinal networks by the cerebellum influences formation of upper limb
16
muscle synergies. This knowledge may support the use of anodal cerebellar DCS as a novel
17
treatment intervention for movement disorders exhibiting abnormal muscle synergy
18
formation such as stroke and dystonia.
19
Page 16
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1
Acknowledgements
2 3
The authors thank Professor Winston Byblow and Associate Professor Cathy Stinear of the University of Auckland for providing the rotor task application for this study.
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
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[18] Alstermark B, Isa T, Tantisira B. Projection from excitatory C3-C4 propriospinal neurones to spinocerebellar and spinoreticular neurones in the C6-Th1 segments of the cat. Neurosci Res. 1990;8:124-30. [19] Alstermark B, Lindström S, Lundberg A, Sybirska E. Integration in descending motor pathways controlling the forelimb in the cat. 8. Ascending projection to the lateral reticular nucleus from C3-C4 propriospinal also projecting to forelimb motoneurones. Experimental brain research. 1981;42:282. [20] Alstermark B, Ekerot C. Organization of the ascending projection from C3–C4 propriospinal neurones to cerebellum via the lateral reticular nucleus. Acta Physiol Scand. 1992;146:P2. [21] Ekerot C-F. The lateral reticular nucleus in the cat. Experimental brain research. 1990;79:109-19. [22] Bradnam L, Young J, Doeltgen S. Anodal Direct Current Stimulation of the Cerebellum reduces Cerebellar-Brain Inhibition but does not influence afferent input from the hand or face in health adults. Cerebellum. 2015;In press. [23] Galea JM, Jayaram G, Ajagbe L, Celnik P. Modulation of cerebellar excitability by polarity-specific noninvasive direct current stimulation. The Journal of Neuroscience. 2009;29:9115-22. [24] Raz N, Williamson A, Gunning-Dixon F, Head D, Acker JD. Neuroanatomical and cognitive correlates of adult age differences in acquisition of a perceptual-motor skill. Microscopy research and technique. 2000;51:85-93. [25] Nicholls ME, Thomas NA, Loetscher T, Grimshaw GM. The Flinders Handedness survey (FLANDERS): a brief measure of skilled hand preference. Cortex. 2013;49:2914-26. [26] Rossini PM, Barker A, Berardelli A, Caramia M, Caruso G, Cracco R, et al. Non-invasive electrical and magnetic stimulation of the brain, spinal cord and roots: basic principles and procedures for routine clinical application. Report of an IFCN committee. Electroencephalography and clinical neurophysiology. 1994;91:79-92. [27] Gandiga PC, Hummel FC, Cohen LG. Transcranial DC stimulation (tDCS): a tool for double-blind sham-controlled clinical studies in brain stimulation. Clinical Neurophysiology. 2006;117:845-50. [28] Rom DM. A sequentially rejective test procedure based on a modified Bonferroni inequality. Biometrika. 1990;77:663-5. [29] Ugawa Y, Uesaka Y, Terao Y, Hanajima R, Kanazawa I. Magnetic stimulation over the cerebellum in humans. Annals of neurology. 1995;37:703-13. [30] Daskalakis ZJ, Paradiso GO, Christensen BK, Fitzgerald PB, Gunraj C, Chen R. Exploring the connectivity between the cerebellum and motor cortex in humans. The Journal of physiology. 2004;557:689-700. [31] Snell RS. Clinical neuroanatomy: Lippincott Williams & Wilkins; 2010. [32] Siegel A, Sapru HN. Essential neuroscience: Lippincott Williams & Wilkins; 2006. [33] Keifer J, Houk JC. Motor function of the cerebellorubrospinal system. Physiological reviews. 1994;74:509-42. [34] Benarroch EE. Basic neurosciences with clinical applications: Elsevier Health Sciences; 2006. [35] Oulad Ben Taib N, Manto M. Trains of epidural DC stimulation of the cerebellum tune corticomotor excitability. Neural plasticity. 2013;2013. [36] Galea JM, Vazquez A, Pasricha N, de Xivry J-JO, Celnik P. Dissociating the roles of the cerebellum and motor cortex during adaptive learning: the motor cortex retains what the cerebellum learns. Cerebral Cortex. 2011;21:1761-70. [37] Bradnam LV, Graetz LJ, McDonnell MN, Ridding MC. Anodal transcranial direct current stimulation to the cerebellum improves handwriting and cyclic drawing kinematics in focal hand dystonia. Frontiers in human neuroscience. 2015;9:286. [38] Schlerf JE, Galea JM, Bastian AJ, Celnik PA. Dynamic modulation of cerebellar excitability for abrupt, but not gradual, visuomotor adaptation. The Journal of Neuroscience. 2012;32:11610-7. [39] Sadnicka A, Patani B, Saifee TA, Kassavetis P, Parees I, Korlipara P, et al. Normal motor adaptation in cervical dystonia: a fundamental cerebellar computation is intact. The Cerebellum. 2014;13:558-67.
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[40] Parazzini M, Rossi E, Ferrucci R, Liorni I, Priori A, Ravazzani P. Modelling the electric field and the current density generated by cerebellar transcranial DC stimulation in humans. Clinical Neurophysiology. 2014;125:577-84. [41] Marchand-Pauvert V, Mazevet D, Pierrot-Deseilligny E, Pol S, Pradat-Diehl P. Handednessrelated asymmetry in transmission in a system of human cervical premotoneurones. Experimental brain research. 1999;125:323-34. [42] DaSilva AF, Volz MS, Bikson M, Fregni F. Electrode positioning and montage in transcranial direct current stimulation. Journal of visualized experiments: JoVE. 2011.
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Figure 1. Schematic diagram showing cerebellar control of putative PNs. The PNs and
2
inhibitory interneurons project to the lateral reticular nucleus, which in turn projects to the
3
cerebellum. Outputs of the cerebellar cortex, via Purkinje neurons provide tonic inhibition of
4
the deep cerebellar nuclei. There are ascending commands to M1 via the dentatothalamic
5
tract and descending commands to PNs indirectly via the corticospinal tract, and brainstem
6
reticulospinal and rubrospinal tracts. These descending pathways modulate excitability of
7
PNs and associated inhibitory interneurons. Not all inhibitory connections are shown for
8
clarity. V shape connections signify excitation, circles signify inhibition. M1, primary motor
9
cortex. T, thalamus. DCN, Deep cerebellar nuclei. RN, red nucleus. RF, reticular formation.
10
LRN, lateral reticular nucleus. PN, propriospinal neuron. αMNs, alpha motoneurons. DTT,
11
dentatothalamic tract. PkN, Purkinje neuron. CST, cerebrospinal tract. RbST, rubrospinal
12
tract. RST, reticulospinal tract. InhN, inhibitory neuron.
13
Figure 2. Average rectified EMG traces of left BB MEPs from 1 representative subject
14
showing FAC and INH, at both pre-DCS (top trace) and post-DCS (bottom trace). NC (grey)
15
and C (black) are shown for FAC and INH at each time point. The MEPAREA was calculated
16
within the time window depicted between the dashed vertical lines for this subject. The ratio
17
of the conditioned to non-conditioned MEPAREA (C/NC) is provided at the right of each trace.
18
Figure 3. A. Pre and post-DCS FAC (black) and INH (grey) MEPs across sham, real and
19
control. Pre-DCS, FAC and INH differed from baseline in all sessions (P < 0.01) apart from
20
sham INH (P = 0.30). Real FAC differed significantly from pre to post-DCS (*P = 0.001).
21
Control INH differed pre to post-DCS (**P = 0.015). Each bar represents the group average
22
(n=12). Error bars indicate SE. B. ΔFAC (black) and ΔINH (grey) across sham, real and
23
control. Positive values indicate an increase in FAC or INH, negative values indicate a
24
decrease. There was a difference in ΔFAC between real and sham (*P = 0.001) as well as real
25
and control sessions (**P = 0.009). Each bar represents the group average (n=12). Error bars Page 20
Page 20 of 27
1
indicate SE. C. ΔSlope across SHAM, real and control. Positive values indicate an increase in
2
Slope, negative values indicate a decrease. Slope did not change significantly between real
3
and sham (P = 0.18) or real and control (P = 0.07). Each bar represents the group average
4
(n=12). Error bars indicate SE.
5
Table 1. Ratios for FAC and INH for the three conditions, pre and post cerebellar DCS and
6
the difference expressed as post – pre. One sample t-tests were used to determine the
7
presence of FAC and INH at baseline. Post hoc paired-sample t-tests were used to assess
8
effects of stimulation on each element. Significance at < 0.05 is signified by * and < 0.001 is
9
signified by †. There was no difference between conditions at baseline for either FAC or INH
10
(all P > 0.11).
11
Table 2. Non-conditioned (NC) MEP (V.ms) and rmsEMG (mV) means ± SE across sham,
12
real and control conditions.
13
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Figure 1
2 3
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1
Figure 2 C NC
0.5mV 10ms
Pre-tDCS FAC
1.66
Pre-tDCS INH
0.70
Post-tDCS FAC
1.14
Post-tDCS INH
0.76
2
3
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1
2 3
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1
Figure 3 **
*
A 1.6
B
1.4
0.1
1.2 *
1
0
∆ C/NC
C/NC
**
*
0.2
0.8 0.6
Sham
Real
Control
-0.1
0.4
-0.2
0.2
-0.3
0 Pre Sham
2
Post
Pre Real FAC
Post
Pre Control
-0.4
Post
FAC
INH
INH
C 0.2 0.1
∆ Slope
0 -0.1
Sham
Real
Control
-0.2 -0.3 -0.4
3 4 5
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1
Table 1 Pre FAC
Post INH
FAC
INH
Difference (Post – Pre) FAC INH
Real
1.40†
0.93*
1.13
0.99
-0.28†
0.07
Control
1.29†
0.94†
1.34
1.05
0.04
0.11
Sham
1.38†
0.97
1.35
1.04
-0.03
0.07
2 3 4
Table 2 NC MEP Pre
NC MEP Post
rmsEMG Pre
rmsEMG Post
SHAM
2.45 ± 0.38
2.77 ± 0.39
0.097 ± 0.036
0.10 ± 0.041
Real
2.35 ± 0.33
2.55 ± 0.19
0.088 ± 0.038
0.091 ± 0.026
Cond
2.68 ± 0.48
2.41 ± 0.33
0.093 ± 0.037
0.094 ± 0.036
SHAM
4.30 ± 0.50
4.73 ± 0.64
0.094 ± 0.033
0.94 ± 0.038
Real
4.14 ± 0.34
4.81 ± 0.41
0.087 ± 0.027
0.095 ± 0.025
Cond
4.88 ± 0.93
4.19 ± 0.94
0.096 ± 0.039
0.092 ± 0.036
FAC
INH
5
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