European Journal of Neuroscience

European Journal of Neuroscience, Vol. 39, pp. 124–131, 2014

doi:10.1111/ejn.12391

NEUROSYSTEMS

Cerebellar continuous theta-burst stimulation affects motor learning of voluntary arm movements in humans Pietro Li Voti,1 Antonella Conte,1,2 Lorenzo Rocchi,2 Matteo Bologna,1 Nashaba Khan,2 Giorgio Leodori2 and Alfredo Berardelli1,2 1

IRCCS Neuromed Institute, Pozzilli, IS, Italy
, 30, 00185 Rome, Italy Department of Neurology and Psychiatry, “Sapienza” University of Rome, Viale dell’Universita

2

Keywords: cerebellum, continuous theta burst stimulation, cortical plasticity, motor learning

Abstract In this study we investigated in healthy subjects whether continuous theta-burst stimulation (cTBS) over the lateral cerebellum alters motor practice and retention phases during ipsilateral index finger and arm reaching movements. In 12 healthy subjects we delivered cTBS before repeated index finger abductions or arm reaching movements differing in complexity (reaching-tograsp and reaching-to-point). We evaluated kinematic variables for index finger and arm reaching movements and changes in primary motor cortex (M1) activity tested with transcranial magnetic stimulation. Peak acceleration increased during motor practice for index finger abductions and reaching-to-grasp movements and persisted during motor retention. Peak acceleration decreased during motor practice for reaching-to-point movements and the decrease remained during motor retention. Cerebellar cTBS left the changes in peak acceleration during motor practice for index finger abductions and reaching-to-grasp arm movements unchanged but reduced peak acceleration at motor retention. Cerebellar cTBS prevented the decrease in peak acceleration for reaching-to-point movements during motor practice and at motor retention. Index finger abductions and arm reaching movements increased M1 excitability. Cerebellar cTBS decreased the motor evoked potential (MEP) facilitation induced by index finger movements, but increased the MEP facilitation after reaching-to-grasp and reaching-to-point movements. Cerebellar stimulation prevents motor retention for index finger abductions, reaching-to-grasp and reaching-to-point movements and degrades motor practice only for reaching-to-point movements. Cerebellar cTBS alters practice-related changes in M1 excitability depending on how intensely the cerebellum contributes to the task. Changes in M1 excitability reflect mechanisms of homeostatic plasticity elicited by the interaction of an ‘exogenous’ (cTBS-induced) and an ‘endogenous’ (motor practice-induced) plasticityinducing protocol.

Introduction Repeating a voluntary motor task induces motor skill learning through two consecutive phases, an early phase followed by a late phase. The early phase consists of a practice-related improvement in motor performance. This achievement – usually measured as an increase in kinematic variables – is retained over a relatively short time (motor retention) and then consolidates after several hours (motor consolidation; Brashers-Krug et al., 1996; Richardson et al., 2006). The late stage of motor learning triggered by further motor practice brings about further gains in motor performance (Karni et al., 1998; Rosenkranz et al., 2007). Studies with transcranial magnetic stimulation (TMS) delivered to the motor cortex show that during practice-related improvement in motor performance motor evoked potential (MEP) amplitude in the trained muscle increases, suggesting that the neural elements in the primary motor cortex (M1) activated by TMS and those

Correspondence: Prof. A. Berardelli, 2Department of Neurology and Psychiatry, as above. E-mail: [email protected] Received 8 February 2013, revised 2 September 2013, accepted 16 September 2013

involved in practice-induced motor memory formation (Stefan et al., 2008; Iezzi et al., 2010; Bologna et al., 2012) at least partially overlap (Muellbacher et al., 2002; Baraduc et al., 2004; Kim et al., 2004; Gregori et al., 2005; Richardson et al., 2006). Repetitive TMS (rTMS) applied to M1 soon after motor practice alters the improvement in motor performance. This finding led some to propose that the primary motor cortex plays a prominent role in early motor learning (Muellbacher et al., 2002; Baraduc et al., 2004). Accordingly, in a study conducted in our laboratory in healthy subjects we showed that continuous theta-burst stimulation (cTBS), a technique that inhibits cortical excitability for up to 60 min (Huang et al., 2005), applied over M1 before motor practice also alters the practice-related improvement in a simple motor task (Iezzi et al., 2010). Unlike data on the inhibitory stimulating protocol, findings on how facilitatory M1 rTMS influences motor learning are controversial (Censor & Cohen, 2011). Although Kim et al. (2004) reported that motor performance during finger tapping improved after facilitatory M1 rTMS, in other experiments similar stimulation protocols left motor learning unchanged (Agostino et al., 2007, 2008). These data overall suggest that excitatory M1 conditioning is probably less effective than inhibitory stimulation

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd

Cerebellum and motor learning 125 in influencing simple motor behaviour in healthy subjects and also indicate that motor learning engages a distributed cortical and subcortical neuronal network. Studies in animals propose that motor learning depends not only on M1 but also on the cerebellum (Ebner, 1998; Doyon et al., 2003, 2009; Yamamoto et al., 2007; Ebner et al., 2011; Manto et al., 2012; Popa et al., 2013). Simple motor learning tasks activate the dentate nuclei and their activation increases as the task becomes more complex (Habas, 2010; K€uper et al., 2011). During early motor task learning, the cerebellum optimizes movement performance through sensory afferent processing, especially for complex movements (Manto et al., 2012). The cerebellar role in motor learning tasks has been recently investigated in humans with TMS and experimental procedures implying contextual interference. Using TMS techniques, Torriero et al. (2004) found that procedural learning is associated with changes in connectivity between the cerebellum and the contralateral motor cortex. The cerebellum modulates cortical excitability during learning acquisition and induces weaker modulation when learning has been acquired. Stimulation applied to the lateral cerebellum can lead to precisional changes in hand movements (Miall & Christensen, 2004; Del Olmo et al., 2007), adaptive motor learning (Galea et al., 2011; Panouill
eres et al., 2012), associative motor learning (Hoffland et al., 2012) and procedural motor learning (Torriero et al., 2004). What remains unclear is whether cerebellar stimulation interferes differentially with motor tasks according to task complexity, thus implying variable degrees of sensorimotor processing (Manto et al., 2012; simple index finger movements, reaching-to-grasp and reaching-to-point arm movements) in the absence of manipulative interventions and whether cerebellar stimulation induces changes in practice-related changes in motor performance as well as in motor retention. Nor is it clear whether possible practice-related changes in motor performance after cerebellar stimulation are associated with changes in M1 excitability. Having clearer background information will help understand the cerebellar role in practice-induced motor memory formation. Our aim in this study was therefore to investigate whether in healthy subjects cTBS applied over the lateral cerebellum can alter practice-related motor practice and motor retention phases during ipsilateral index finger abductions and reaching movements involving the arm. To do so we delivered cTBS over the lateral cerebellum immediately before subjects engaged in motor practice involving simple index finger abductions and arm reaching movement tasks that differed in complexity (reaching-to-grasp and reaching-to-point an object). To see whether cerebellar-induced changes in motor performance take place through changes in M1 cortical plasticity mechanisms we used single-pulse TMS to monitor M1 cortical activity.

Materials and methods Twelve right-handed healthy subjects [six men, six women; mean ( SD) age 30.2  5.78 years] participated in the study after giving written informed consent. The experimental procedures were approved by the institutional review board of Sapienza University of Rome, and conducted in accordance with the Declaration of Helsinki and the international TMS guidelines (Rossi et al., 2009). Transcranial magnetic stimulation A monophasic Magstim stimulator (Magstim SuperRapid; The Magstim Company Ltd, Whitland, UK) connected to a figure-of-eight coil was used to deliver single TMS pulses over the first

dorsal interosseous muscle (FDI) motor hot-spot on the left hemisphere (to probe M1 excitability after right lateral cerebellum cTBS). Cerebellar cTBS was delivered with a Super Rapid Magstim 200 stimulator (The Magstim Company Ltd). The coil was positioned tangentially to the scalp, with the handle pointing superiorly. cTBS was delivered over the right cerebellar hemisphere with the coil placed 1 cm inferior and 3 cm to the left of the inion. In accordance with previous studies, the stimulation scalp site we used corresponds to the posterior and superior lobules in the lateral cerebellum (Del Olmo et al., 2007; Koch et al., 2007) and the coil orientation used allowed us to modulate contralateral hand motor area excitability (Oliveri et al., 2005; Koch et al., 2008). Bursts of three pulses at 80% active motor threshold (AMT) were delivered at 50 Hz and repeated every 200 ms in a continuous train lasting 40 s for a total 600 pulses (Huang et al., 2005; Iezzi et al., 2008; Conte et al., 2012a,b). To determine the intensity of cerebellar cTBS a figure-ofeight coil (external wing 9 cm in diameter) was placed over the right M1 with the handle pointing back and away from the midline at about 45°, in the optimal position (hot spot) for eliciting MEPs in the left FDI muscle. AMT was calculated during a 20–30% maximum voluntary contraction in the target muscle as the lowest intensity able to evoke an MEP of at least 200 lV in five of 10 consecutive trials. Electromyographic recordings Electromyographic data and MEPs were recorded through a pair of Ag/AgCl electrodes placed over the right FDI muscle (to test AMT) and left FDI muscle (to test M1 excitability before and after cTBS) in a belly-tendon fashion. Raw signal, sampled at 5 kHz with a CED 1401 A/D laboratory interface (Cambridge Electronic Design, Cambridge, UK), was amplified and filtered (bandwidth 20–1 kHz) with a Digitimer D 360 (Digitimer Ltd, Welwyn Garden City, UK). Data were stored on a laboratory computer for on-line visual display and further off-line analysis (Signal software; Cambridge Electronic Design). Movement recording and motor tasks The SMART analyser motion system (BTS Engineering, Milan, Italy), equipped with three infrared cameras (sampling rate, 120 Hz), was used to record index finger abductions and arm reaching movements. During the motor task subjects were comfortably seated in an armchair beside a table. For the index finger movements the right arm was firmly secured and the arm position was kept constant throughout the experiment by visually inspecting the joint angles and by keeping the distance between the armchair and the table stable throughout the experiments. Care was taken to secure the right forearm firmly in the same position on the table. The right arm was abducted at the shoulder by about 45–50° and the elbow joint was flexed at about 90° (Agostino et al., 2007). An optical marker was placed over the distal phalanx of the dominant index finger. Marker displacement was reconstructed via dedicated software running the automatic algorithm to compute acceleration peak values. After a verbal ready signal, subjects were asked to extend their index finger until they reached the neutral position at the metacarpo-phalangeal joint. Then, after a verbal ‘go’ signal subjects abducted the index finger and soon after a verbal ‘stop’ signal they returned the finger to the starting position (Agostino et al., 2007; Li Voti et al., 2011; Bologna et al., 2012). Subjects were asked to abduct the index finger as accurately, as widely and as fast as possible and were continuously encouraged to do so

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 124–131

126 P. Li Voti et al. throughout the motor task. We measured movement amplitude and peak acceleration. For arm reaching movements, using their thumb and index finger subjects were instructed to reach precisely and grasp as fast as possible a small (2 cm in diameter) cylindrical body (~15 cm above the table at sternal height). The cylindrical body was placed on the table at two-thirds arm’s length from the body midline. The endpoint marker was placed on the styloid apophysis of the radius. To make arm reaching movements more complex we used a motor task characterized by muscle groups activated during the task and target distance (two-thirds arm’s length away from the body midline) similar to the reaching-to-grasp movements but requiring higher accuracy. A thin plastic cylinder (1 mm in diameter) was mounted on the right index finger and subjects were instructed to reach a small target (a bulls eye 2 mm in diameter) and insert the plastic cylinder into the bulls eye as precisely and as fast as possible. The endpoint marker was placed on the styloid apophysis of the radius. For arm reaching movements we recorded kinematic measures for performance (i.e. peak acceleration, duration, acceleration and deceleration phase duration) and movement quality [i.e. trajectory characteristics – trajectory straightness determined by the index of curvature (IC), an index assessing the path curvature from the initial position to target contact expressed as the ratio of the curved path length to the straight-line distance between the initial and final positions, both computed in three-dimensional space (Deuschl et al., 2000). The endpoint trajectory smoothness was measured as the number of peaks (movement units) in the endpoint velocity curve. A movement unit was defined as a local maximum velocity preceded by increasing values and followed by decreasing values, for at least 20 ms.] Movement duration was measured as the time elapsing between movement onset to the moment when subjects completely inserted the plastic cylinder into the bulls eye. After a brief practice trial, 200 index finger abductions/reachingto-grasp/reaching-to-point movements were executed in blocks of 20 movements. One hour after motor practice ended, subjects did 20 further movements to test motor retention. To avoid fatigue, a 3-s interval elapsed between movements and a 15-s interval between blocks. The kinematic variables were calculated for each movement and then averaged for each block. Experimental paradigm Each subject underwent seven experimental sessions – three involving motor practice for index finger abductions/reaching-to-grasp movements/reaching-to-point movements, three further sessions consisting in motor practice preceded by cerebellar cTBS (cerebellar cTBS and index finger abductions/cerebellar cTBS and reaching-to-grasp movements/cerebellar cTBS and reaching-to-point movements), and a session with cerebellar cTBS alone. Sessions with and without cTBS were randomized and counterbalanced across the subjects. At least 1 week elapsed between each experimental session. In each session we determined the single TMS pulse intensity able to evoke at least 10 consecutive MEPs at about 1 mV peak-to-peak amplitude at baseline (T0). The same intensity was used for testing MEP size 15 min (T1), 30 min (T2) and 60 min (T3) after cTBS and, for the experiments with index finger and reaching movements, 60 min after the motor task ended, a time corresponding to motor retention assessment (Fig. 1). Twenty MEPs were averaged at each time point. To ensure that subjects completely relaxed the target muscle during MEP assessment and during cTBS, the investigators continuously monitored electromyographic activity with audio and high-gain visual feedback.

Fig. 1. Experimental paradigm – each subject underwent seven experimental sessions, three involving motor practice for index finger abductions/reachingto-grasp movements/reaching-to-point movements, three further sessions consisting of motor practice preceded by cerebellar cTBS (cerebellar cTBS and index finger abductions/cerebellar cTBS and reaching-to-grasp movements/ cerebellar cTBS and reaching-to-point movements), and a session with cerebellar cTBS alone. In each session MEPs were recorded before (T0) and 15 (T1), 30 (T2) and 60 (T3) minutes after cTBS.

Statistical analysis Because the instructions subjects received differed for each motor task we used separate within-subjects repeated measures analysis of variance (ANOVAs) with factor CONDITION (two levels – movement alone vs. movement preceded by cerebellar cTBS) and BLOCK (11 levels – 10 blocks for motor practice and one block for motor retention) to analyse changes in kinematic variables for each of the three movements. ANOVAs were tested for non-sphericity with Mauchly’s test and the Greenhouse–Geisser correction for non-sphericity was applied when needed. A paired-sample t test was used to analyse changes in kinematic variables for each block compared with the first block. Within-subjects repeated measures ANOVA with factor EXPERIMENTAL SESSION (seven levels – index finger alone, index finger preceded by cerebellar cTBS, simple reaching alone and simple reaching preceded by cerebellar cTBS, complex reaching alone and complex reaching preceded by cerebellar cTBS and cTBS alone) and TIME (four levels: T0, T1, T2, T3) was used to analyse changes in MEP amplitude. Separate repeated measures ANOVAs were run for post hoc analysis to identify changes in MEP size in the ‘movement alone’ vs. ‘cerebellar cTBS and movements’ conditions with TIME (four levels: T0, T1, T2, T3) and CONDITION (two levels – movements alone vs. cerebellar cTBS and movements). Post hoc analysis to disclose whether the changes in the MEP size induced by motor practice differed according to movement type was also used with factor TIME and MOVEMENT TYPE as main factors. A paired sample t-test were also used for analysing when the most significant changes in MEP size took place. Correlations between TMS and kinematic variables were assessed by calculating Pearson’s correlation coefficient. Holm’s correction for multiple comparison was used to disclose false significance. P-values ≤ 0.05 were considered to indicate statistical significance. Data are shown as mean  SE.

Results Index finger and reaching arm movements – cerebellar cTBSinduced effects on kinematic variables Index finger movements Within-subjects repeated-measure ANOVA for peak acceleration of index finger movements showed a significant effect of factor BLOCK (F10,110 = 6.01, P < 0.0001) and a significant interaction of

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 124–131

Cerebellum and motor learning 127

Fig. 2. Changes in peak acceleration during the motor practice task testing index finger movements alone and index finger movements preceded by cerebellar continuous theta-burst stimulation (cTBS). Each column represents mean value, and bars represent standard error.

Fig. 3. Changes in peak acceleration during the motor practice task testing reaching-to-grasp movements alone and reaching-to-grasp movements preceded by cerebellar continuous theta-burst stimulation (cTBS). Each column represents mean value, and bars represent standard error.

factor BLOCK and CONDITION (F10,110 = 2.23, P = 0.02). Post hoc tests for changes in peak acceleration during the index finger experiment showed that peak acceleration increased during the motor task (F10,110 = 4.64, P = 0.00001) and the increase was significant at block VIII (P = 0.003), IX (P = 0.0008), X (P = 0.0008) and at motor skill retention assessment (P = 0.003). Conversely, when cerebellar cTBS preceded index finger movements, peak acceleration increased during the motor task (F10,110 = 4.09, P = 0.00008) and the increase was significant at block VII (P = 0.006), VIII (P = 0.01), IX (P = 0.02) and X (P = 0.03) but not at motor retention assessment (P = 0.97). Within-subjects repeated-measure ANOVA for movement amplitude during index finger movements showed a significant effect of factor BLOCK (F10,110 = 5.01, P = 0.02) but no significant effect of factor CONDITION (F10,110 = 0.02, P = 0.89) or interaction between the factors BLOCK and CONDITION (F10,110 = 0.04, P = 0.95). Cerebellar cTBS therefore prevented motor skill retention for the increase in peak acceleration (Fig. 2).

Reaching-to-point movements

Reaching-to-grasp movements Within-subjects repeated-measure ANOVA for peak acceleration showed a significant interaction of factor BLOCK and CONDITION (F10,110 = 2.10, P = 0.02) but no effect of factor BLOCK (F10,110 = 0.84, P = 0.58) and CONDITION (F10,110 = 0.04, P = 0.83). Post hoc tests for peak acceleration during motor retention vs. peak acceleration at block I in the two conditions showed a significant interaction between factor BLOCK and CONDITION (F1,11 = 7.48, P = 0.01); when subjects made movements alone, peak acceleration was higher at motor retention assessment than in block I (paired sample t-test comparing peak acceleration at the Ist block vs. retention block – P = 0.01), but remained unchanged when cerebellar cTBS preceded reaching-to-grasp movements (paired sample t-test comparing peak acceleration at block I vs. retention block – P = 0.36; Fig. 3). Smoothness, IC, deceleration and acceleration phase duration remained statistically unchanged (Table 1). Cerebellar cTBS therefore prevented motor skill retention for the increase in peak acceleration but left smoothness, IC, deceleration and acceleration phase durations unaffected.

Within-subjects repeated-measure ANOVA for peak acceleration showed a significant effect of factor BLOCK (F10,110 = 5.42, P = 0.0000001) and interaction between factor BLOCK and CONDITION (F10,110 = 1.90, P = 0.04) but no effect of factor CONDITION (F10,110 = 3.90, P = 0.08). Post hoc tests showed that peak acceleration changed significantly when subjects did the motor task alone (F10,110 = 5.87, P < 0.00001) but remained unchanged when cerebellar cTBS preceded movements (F10,110 = 1.79, P = 0.08). A post hoc t-test comparing peak acceleration for each movement blocks vs. I block in the two conditions showed that peak acceleration decreased during the motor practice and the decrease was significant at blocks VI (P = 0.003), VII (P = 0.001), VIII (P < 0.0001), IX (P < 0.0001) and X (P < 0.0001), and at motor retention (P = 0.01) when subjects did reaching-to-point movements alone whereas it was non-significant when cerebellar cTBS preceded the motor task (Fig. 4). Acceleration phase duration increased durTable 1. Statistical values of smoothness, curvature index, acceleration and deceleration phase duration of reaching-to-grasp and reaching-to-point movements Reaching-to-grasp

Reaching-to-point

F

F

Smoothness Block 0.22 Condition 0.03 Block 9 Condition 0.85 Curvature index Block 0.83 Condition 2.32 Block 9 Condition 0.36 Acceleration phase duration Block 0.24 Condition 0.03 Block 9 Condition 1.20 Deceleration phase duration Block 0.95 Condition 0.0001 Block 9 Condition 0.3

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 124–131

P-values d.f.

P-values d.f.

0.99 0.84 0.57

10,110 0.71 1,11 0.32 10,110 0.80

0.71 0.58 0.62

10,110 1,11 10,110

0.59 0.15 0.96

10,110 0.73 0.68 1,11 0.0004 0.98 10,110 1.15 0.33

10,110 1,11 10,110

0.99 0.84 0.29

10,110 3.74 1,11 0.35 10,110 1.36

0.0002 0.56 0.20

10,110 1,11 10,110

0.48 0.99 0.97

10,110 1.38 1,11 4.85 10,110 1.46

0.20 0.04 0.16

10,110 1,11 10,110

128 P. Li Voti et al. Table 2. Statistical values of changes in MEP size

Index finger movements Experimental session Time Experimental session 9 Time Post hoc movement alone Post hoc cTBS + mov. Reaching-to-grasp movements Experimental session Time Experimental session 9 Time Post hoc movement alone Post hoc cTBS + mov. Reaching-to-point movements Experimental session Time Experimental session 9 Time Post hoc movement alone Post hoc cTBS + mov. Fig. 4. Changes in peak acceleration during the motor practice task testing reaching-to-point movements alone and reaching-to-point movements preceded by cerebellar continuous theta-burst stimulation (cTBS). Each column represents mean value, and bars represent standard error.

ing the blocks and did so to a similar extent in the two conditions (Table 1). Deceleration phase duration increased during the blocks but the deceleration phase duration was lower when the subjects did movements alone than when cerebellar cTBS preceded complex reaching movements. Smoothness and IC remained significantly unchanged (Table 1). In the reaching-to-point movements, subjects improved their movement performance accuracy by reducing peak acceleration and increasing acceleration and deceleration phase durations. Cerebellar cTBS altered practice-related motor memory formation by preventing changes in peak acceleration and in deceleration phase duration during motor practice and at the motor retention assessment.

F

d.f.

P-values

0.31 4.75 5.63 6.67 2.73

1,11 3,33 3,33

0.58 0.007 0.003 0.001 0.06

2.83 6.20 5.18 3.10 7.68

1,11 3,33 3,33

0.12 0.001 0.004 0.03 0.0005

0.31 6.95 0.52 4.12 3.76

1,11 3,33 3,33

0.58 0.0009 0.66 0.01 0.02

reaching-to-point movements, the MEP increase was larger when cerebellar cTBS preceded the motor task (Fig. 5, Table 2). MEP size increased more during index finger movements alone than during arm reaching movements (TIME: F3,33 = 9.33, P = 0.0001; TIME and MOVEMENT TYPE interaction: F3,33 = 3.58, P = 0.003). No difference was found for MEPs at T0 in the seven experimental sessions (F6,66 = 0.67, P = 0.67). Correlations between changes in M1 excitability and changes in kinematic variables Pearson’s correlation analysis showed no significant correlation between changes in MEP size and changes in kinematic variables for the three types of movements.

Discussion Cerebellar cTBS and motor practice – changes in M1 excitability Within-subjects repeated-measure ANOVA for MEP amplitude showed a significant effect of factor TIME (F3,33 = 13.29, P = 0.0000007) and a significant interaction between factor EXPERIMENTAL SESSION and TIME (F18,198 = 3.30, P = 0.000001) but no effect of factor EXPERIMENTAL SESSION (F6,66 = 1.91, P = 0.09). Post hoc analysis showed that MEP amplitude significantly decreased after cerebellar cTBS alone (F3,33 = 5.63, P = 0.003) and the decrease was significant at T1 (P = 0.0002) and T2 (P = 0.04) and returned to baseline values at T3 (P = 0.5). MEP significantly increased after index finger movements alone and the increase was significant at T1 (P = 0.0007) and T2 (P = 0.005) whereas it increased but not significantly when index finger movements were preceded by cTBS (Table 2). In the reaching-to-grasp sessions, MEP increased after movements alone (significantly at T1 – P < 0.001) and also when cTBS preceded reaching-to-grasp movements (T2 – P = 0.006, T3 – P = 0.01). Similarly, in the reaching-to-point movements sessions, MEP size increased when movements were done alone (T2 – P = 0.01) and also when reaching movements were preceded by cTBS (T2 – P = 0.004, T3 – P = 0.02). Cerebellar cTBS therefore differentially modulated MEP size depending on the movement type tested. The MEP increase in size when subjects did index finger movements alone was larger than that observed when cerebellar cTBS preceded the motor task. When subjects did reaching-to-grasp and

In this study, we show that during repeated index finger abductions and reaching-to-grasp movements peak acceleration increases and the increase persists at the motor retention phase whereas during reaching-to-point movement peak acceleration progressively decreases during the task and also during the motor retention phase. Cerebellar cTBS leaves the practice-related changes in peak acceleration unchanged for index finger movements and for reaching-tograsp movements whereas it influences changes in peak acceleration for reaching-to-point movements. We also show that cerebellar cTBS modifies peak acceleration at the motor retention phase for all the three movement types. Conversely, the smoothness and straightness in trajectories related to arm reaching movements remain unchanged. When subjects repeat index finger and arm reaching movements, M1 excitability increases and the increase is higher during index movements than during arm reaching movements. Cerebellar cTBS influences movement-related changes in M1 excitability in two ways – it decreases the MEP facilitation induced by index finger movements but increases the MEP facilitation induced by arm reaching movements. No correlation was found between changes in peak acceleration and MEP size at the motor retention assessment. These new findings imply that cerebellar stimulation in healthy subjects prevents motor retention for index finger abductions and for both arm reaching movements tested but also degrades motor practice for arm reaching movements that increase in difficulty (reaching-to-point task). The cerebellar mechanisms underlying changes in

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 124–131

Cerebellum and motor learning 129

Fig. 5. Changes in motor evoked potential (MEP) amplitude during the motor practice of index finger movements (upper panel), reaching-to-grasp (middle panel) and reaching-to-point movements (lower panel) alone and motor tasks preceded by cerebellar continuous theta-burst stimulation (cTBS). Each point represents mean value, and bars represent standard error.

M1 excitability and motor learning therefore differ according to motor task complexity. We took several precautions to ensure reliable findings. The within-subjects design in our experimental procedures ensured that our differential findings on M1 excitability and motor learning were unaffected by possible differences in the subjects enrolled for index finger and reaching movements. Because subjects were randomly assigned to begin with the experimental session testing movements alone vs. cerebellar cTBS and movements we also feel confident that we excluded an order effect. We also exclude a ‘take over’ effect due to the multisession design given that experimental sessions took place at least 1 week apart and the MEP size measured at baseline remained unchanged in all the experimental sessions. Because cerebellar cTBS influenced MEP size we feel confident that the lack of changes in kinematic variables during motor practice for index finger movements and during reaching-to-grasp movements did not depend on ineffective stimulation. Further evidence showing an effective stimulation protocol came from changes in peak acceleration of index finger and arm reaching movements at the motor retention assessment concomitantly to changes in MEP size. Excluding all technical confounders, the lack of changes in the kinematic variables during the motor practice phase for index and reaching-to-grasp movements after cerebellar cTBS might therefore reflect the prominent role of M1 in the early motor learning phases (Muellbacher et al., 2002; Baraduc, 2004). Support for this explanation comes from the observation that training on a novel skill produces changes in the neural circuitry in the motor cortex that are

specific to the muscle groups necessary for executing the trained task (Adkins et al., 2006). Cerebellar cTBS, however, altered practicerelated changes in peak acceleration in the reaching-to-point movements. The decrease in peak acceleration when subjects practised reaching-to-point movements shows that the task is more complex than the task testing index finger abductions and reaching-to-grasp movements. Hence the extent to which cerebellar cTBS influences motor practice probably depends on movement task complexity. The changes in peak acceleration we observed during motor retention for all movement types suggest that cerebellar cTBS alters the mechanisms involved in motor memory formation. Studies in animals consistently show that motor learning and consolidation both require distributed plasticity in the granule-cell network and Purkinje-cell networks. Plasticity in the granule-cell network may increase coding diversity, whereas plasticity in the Purkinje-cell network may facilitate selecting the appropriate coding and transferring it to the output domain that controls the appropriate movement. Studies in animals also demonstrated that cerebellar cortex activity taking place in the post-training phase is important for motor memory (Kassardjian et al., 2005; Shutoh et al., 2006; Kellett et al., 2010; Okamoto et al., 2011; Gao et al., 2012). Hence in the healthy subjects we studied cerebellar cTBS might have determined changes in the cerebellar cortex activity responsible for motor memory formation. Our findings partly contrast with those from Galea et al. (2011), who found that anodal cerebellar transcranial direct current stimulation (tDCS) specifically enhanced acquisition without influencing motor retention. The differences could arise from numerous factors – the motor learning task used in their study tested adaptive motor learning to a novel visuomotor transformation whereas our motor learning task tested the practice-related mechanisms underlying motor memory formation. And possibly more importantly, the two techniques differ also insofar as Galea et al. (2011) used facilitatory tDCS whereas we used inhibitory TBS. Experimental stimulation protocols designed to interfere with cerebellar activity determine changes in the acquisition phase or motor retention phases depending on how much the cerebellum contributes in the motor tasks tested. Our observation that cerebellar cTBS altered peak acceleration during arm reaching movements but left movement smoothness and trajectory unchanged suggests that movement acceleration and trajectory control depend on separate functional channels (Milak et al., 1997; Cooper et al., 2000; Martin et al., 2000; Manto et al., 2012). Supporting our hypothesis that movement velocity and trajectory control involve separate functional pathways in the cerebellum, animal studies investigating reaching movements showed that dentatus nucleus inactivation elicited significant movement slowing but did not degrade performance as measured by trajectory (Beaubaton & Trouche, 1982; Martin et al., 2000). Cerebellar cTBS might have interfered with motor retention by inhibiting M1 activity. Although this explanation concords with previous reports suggesting that M1 has a role in retaining new motor memories (Muellbacher et al., 2002; Richardson et al., 2006; Hadipour-Niktarash et al., 2007; Galea & Celnik, 2009; Hunter et al., 2009; Reis et al., 2009) the lack of correlation between changes in M1 excitability and changes in kinematic variables during motor retention for index finger and arm reaching movements suggests that more complex mechanisms underlying cerebellar cTBS-induced effects intervene. Cerebellar cTBS differentially influenced M1 excitability depending on whether subjects did index finger or reaching movements before or after receiving cTBS. One possibility to explain why cerebellar cTBS stimulation inhibited the MEP increase after index finger movements is that cortical neurons activated by TBS are ‘occluded’ and therefore unresponsive to subsequent motor learning processes. Con-

© 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, 39, 124–131

130 P. Li Voti et al. versely, the cerebellar cTBS-induced higher MEP increase after both types of reaching arm movements we studied deserves a different explanation. Several studies (Iyer et al., 2003; Lang et al., 2004; Siebner et al., 2004) showed that after priming M1 with a long-term depression (LTD)-inducing protocol, subsequent information induced on the same cortical area can reverse the expected after-effects. More recently, Popa et al. (2013) showed that TBS over the cerebellum alters the motor cortex response to the various plasticity-inducing protocols according to the presence or absence of sensory afferent components through mechanisms involving homeostatic plasticity. LTD–long-term potentiation (LTP)/like induced plasticity depends on previous synaptic activity. The threshold for LTP/LTD induction after a stimulation protocol depends especially on the integrated postsynaptic activity termed ‘homeostatic synaptic plasticity’ (Abraham & Tate, 1997; Abbott & Nelson, 2000; Davis, 2006). Because the arm reaching movements we studied require more proprioceptive information and a more prominent cerebellar contribution than do index finger abductions (Manto et al., 2012), the increased MEP facilitation we found when we applied cerebellar cTBS before subjects did reaching movements could arise through homeostatic plasticity mechanisms. We propose that owing to proprioceptive information processing, the prominent cerebellar activation during reaching movements primed by an inhibitory cerebellar stimulation protocol (cTBS-related LTD-like effects) reverts the cerebello-thalamic influences to M1 from inhibition (Koch et al., 2008) to facilitation. Homeostatic interactions between cTBS-related LTD-like processes and reaching movements-related cerebellar activation therefore primarily intervene at cerebellar level and indirectly determine remote changes in M1 excitability. Cerebello-thalamic influences to M1 probably contribute to shaping M1 activity depending on whether subjects are tested at rest or during muscle activation. This possibility notwithstanding, our finding that cerebellar cTBS given before arm reaching movements enhances M1 excitability whereas before index finger movements it does not implies that the homeostatic interaction between cTBS-induced effects and LTP-like plasticity processes underlying repeated reaching movements depends specifically on movement complexity (i.e. the prominent cerebellar role). Further supporting this hypothesis, cerebellar cTBS determines a higher increase in MEP size in the more highly complex reaching-to-point tasks than in the reaching-to-grasp movements at the motor retention phase. The observation that cTBS given before reaching-to-grasp and reaching-to-point movements elicited a larger MEP than index finger movements and elicited opposite changes in peak acceleration during motor practice (peak acceleration increases during reaching-to-grasp whereas it decreases during reaching-topoint movements) suggests that changes in M1 excitability are not specifically related to changes in peak acceleration. As FDI muscle is less prominently activated during reaching movements than during index finger abductions, testing M1 excitability from FDI muscle might be not completely representative of changes in the excitability of cortical areas controlling muscles (i.e. biceps, triceps, forearm muscles) more specifically involved in reaching tasks. However, considering that FDI muscle is also activated during reaching arm movements, although to a lesser extent in comparison to the index finger movements, testing M1 excitability from FDI muscle probably only underestimates the effects of cerebellar modulation on M1. Future research testing cerebello-thalamocortical pathways with paired-pulse TMS (Ugawa et al., 1995) might disclose more specific M1 excitability changes. In conclusion, no matter how complex the motor task tested, testing practice-related improvement in kinematic variables during index finger and reaching arm movements, cerebellar cTBS interferes with

motor retention. By increasing motor task complexity and consequently augmenting the possible cerebellar role, cerebellar cTBS may interfere also with motor practice. cTBS alters motor practicerelated synaptic activity in M1, by occluding M1 activity for index finger movements and by inducing homeostatic plasticity mechanisms for arm reaching movements.

Acknowledgements No funding was received. The authors declare no competing interests.

Abbreviations AMT, active motor threshold; ANOVA, analysis of variance; cTBS, continuous theta-burst stimulation; FDI, first dorsal interosseous muscle; IC, index of curvature; M1, primary motor cortex; MEP, motor evoked potential; rTMS, repetitive TMS; TMS, transcranial magnetic stimulation.

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