Clinical Neurophysiology 126 (2015) 1016–1023

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Differential effects of facilitatory and inhibitory theta burst stimulation of the primary motor cortex on motor learning Milan B. Jelic´, Sladjan D. Milanovic´, Saša R. Filipovic´ ⇑ University of Belgrade, Institute for Medical Research, Department of Neurophysiology, Belgrade, Serbia

a r t i c l e

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Article history: Accepted 3 September 2014 Available online 16 September 2014 Keywords: Motor learning Primary motor cortex Transcranial magnetic stimulation Neuromodulation Plasticity

h i g h l i g h t s  If applied during early consolidation phase of learning a complex motor task, theta burst stimulation

(TBS) can alter the learning process.  Along with a significant drop in primary motor cortex (M1) excitability, inhibitory continuous TBS

(cTBS) induces sustained and significant slowing of motor learning.  TBS induced M1 excitability change and magnitude of motor learning during early consolidation

phase correlate significantly.

a b s t r a c t Objective: To evaluate the differential effects on motor learning of two types of theta burst stimulation (TBS), the excitatory intermittent TBS (iTBS) and inhibitory continuous TBS (cTBS), if TBS is applied in an early stage of learning process. Methods: Thirty right handed healthy people were randomly allocated into one of the three groups according to the intervention applied, iTBS, cTBS or placebo. The interventions and measurements targeted the non-dominant side. The reaction time task (RTT) and Purdue pegboard task (PPT) were used. Measurements and motor tasks were carried out at baseline (T0), immediately after the intervention (T1), and 30 min later (T2). Results: Compared to placebo, following cTBS M1 excitability went down and PPT learning was slowed. Following iTBS M1 excitability increased temporarily and PPT learning pattern changed, but learning was not improved. The MEP and PPT changes induced during the T0–T1 time interval correlated significantly. Conclusions: The early consolidation of the learned material was much more influenced by the TBS induced promotion/suppression of the M1 functional plasticity reserves than by the absolute level of the M1 activation. Significance: The results may help to better define the use of TBS in promotion of motor learning in neurorehabilitation and cognitive enhancement. Ó 2014 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.

1. Introduction Motor learning involves three basic stages: the early stage of rapid learning, the later stage of slower improvement of a motor skill performance, and the final stage of development of automaticity in performance, which occurs when the skill is completely learned (Halsband and Lange, 2006). Within the early motor ⇑ Corresponding author at: Department of Neurophysiology, UB Institute for Medical Research, P.O. Box 39, 11129 Beograd 102, Serbia. Tel.: +381 11 2685788x104; fax: +381 11 2643691. E-mail address: sasa.fi[email protected] (S.R. Filipovic´).

learning stage, two alternating phases can be distinguished: acquisition and early consolidation. The acquisition phase involves within-session performance improvements (‘‘on-line learning’’) which appear during actual task/skill practice at the initial learning sessions. The early consolidation phase involves further performance improvements that take place between acquisition sessions, without actual training (‘‘off-line learning’’) (Krakauer and Shadmehr, 2006; Tanaka et al., 2011).1

1 It has to be distinguished from the late consolidation which refers to retention of motor performance after several hours from the end of motor practice.

http://dx.doi.org/10.1016/j.clinph.2014.09.003 1388-2457/Ó 2014 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.

M.B. Jelic´ et al. / Clinical Neurophysiology 126 (2015) 1016–1023

A number of cortical and subcortical structures are active during early motor learning (Shadmehr and Holcomb, 1997; Hardwick et al., 2013). However, the activity in the primary motor area (M1) seems to be of a specific significance during this stage (Pascual-Leone et al., 1995; Nudo et al., 1996; Classen et al., 1998; Kleim et al., 1998; Plautz et al., 2000; Muellbacher et al., 2001). Several animal (Rioult-Pedotti et al., 1998, 2000) and human (Ziemann et al., 2004; Iezzi et al., 2008; Jung and Ziemann, 2009) studies highlighted the interaction between motor learning and long-term potentiation (LTP) and long-term depression (LTD)-like synaptic plasticity in M1. Noninvasive neuromodulatory methods based on transcranial magnetic stimulation (TMS) can efficiently change M1 excitability and induce either LTP or LTD-like effects (Hoogendam et al., 2010). The brevity of its application and strong theoretical link with basic mechanisms of neuronal interactions make one of these methods, the theta burst stimulation (TBS) (Huang et al., 2005), particularly suitable for human studies of M1 involvement in motor learning. This is especially the case for potential further use of the method in rehabilitation of motor impairments following stroke and other brain injuries. However, so far only a handful of studies have investigated the relationships between TBS induced M1 plasticity and motor learning and in each of those studies only a single type of TBS was used, either one inducing LTP-like effect or the one inducing LTD-like effect (Agostino et al., 2008; Iezzi et al., 2010; Teo et al., 2011). This relative paucity of data, together with incongruent results obtained in some of the studies, does not allow any conclusions to be made regarding the effects of TBS induced M1 plasticity and motor learning. Moreover, the tasks studied were typically rather simple (fast finger abductions) which do not allow for direct transfer of the obtained results to the realistic real-life situations where more complex skills have to be learned. In this placebo-controlled parallel-groups study, we compared the effects of the two TBS types on motor learning in healthy participants. The effects of the intermittent TBS (iTBS), which typically induces the LTP-like plasticity, were compared with the effects of the continuous TBS (cTBS), which was shown to induce the LTDlike plasticity (Huang et al., 2005). All participants performed the same two motor tasks, one simple and another complex, and TBS interventions were applied to M1 at the same point in the learning process, the point which corresponded with the early consolidation phase. The design of the study thus allowed for direct comparisons of the effects the two TBS types. It has been shown that dominant hemisphere and its M1 are not only responsible for control of the contralateral hand, but have significant role in higher-level control of motor learning with either of the hands (Grafton et al., 1995, 2002; Schambra et al., 2011). In contrast, the non-dominant hemisphere seems to be involved in control of the contralateral hand exclusively (Serrien and Spape, 2009). Hence, we considered non-dominant M1 to be better model for evaluating of the low-level M1 motor learning processes and, in this study, focused our attention to this area. Moreover, nondominant M1 with its lack of specialization and proficiency in higher-level motor learning processes (Schambra et al., 2011) may have features resembling of a damaged M1 (e.g. after stroke, trauma) allowing thus drawing conclusions that could be relevant for neurorehabilitation as well.

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participants were randomly allocated into one of the three groups: two experimental groups, the iTBS group and the cTBS group (mean age: 25 ± 3.4 [6 men] and 26 ± 2.5 [7 men], respectively), which were named in accordance with the type of the experimental intervention applied, and the placebo group (26 ± 1.9 [5 men]), which received sham stimulation. All participants gave written informed consent, following the Declaration of Helsinki; the study protocol was approved by the local Ethics Committee. 2.2. Stimulation and recording techniques All investigations were performed on the non-dominant left hand with participants comfortably seated in a chair. The electromyography (EMG) data were collected from the first dorsal interosseus muscle (FDI) using Ag–AgCl electrodes placed in a belly-tendon montage. EMG activity was amplified (1000), filtered (10 Hz–1 kHz) and then sampled at 2 kHz (CED 1401 plus, Cambridge Electrical Design, Cambridge, UK). The data were stored on a computer for off-line analysis. All stimulation was carried out using a Magstim Rapid stimulator (Magstim Co., Whitland, Wales, UK) and the 70-mm figure-of-eight coil. The handle of the coil was oriented to a direction posterior to the midline at a 45° angle in order to allow the electromagnetic currents to flow perpendicular to the central sulcus. 2.3. Transcranial magnetic stimulation (TMS) measurements The optimal scalp location (‘‘hot spot’’) for FDI stimulation was determined using single-pulse TMS. Subsequently, the resting motor threshold (RMT) was determined as the lowest stimulus intensity necessary to produce motor-evoked potentials (MEPs) of peak-to-peak amplitude P50 lV in 5 of 10 subsequent trials (Rossini et al., 1994). Finally, the active motor threshold (AMT) was determined as the lowest stimulus intensity sufficient to produce MEP of peak-to-peak amplitude P100 lV in 5 of 10 subsequent trials during weak voluntary activation of the FDI. The TMS pulses of 120% RMT intensity were used to evoke a MEP in relaxed FDI to assess the M1 excitability. Muscle relaxation was monitored and subjects had the visual feedback of their EMG. Ten MEPs were recorded at each assessment time-point and the peakto-peak amplitudes were averaged. The trials in which background EMG activity was present were excluded from the averages. 2.4. Intervention The intervention consisted of two types of TBS. In both of them, bursts of TMS pulses (80% AMT intensity) were applied at 5 Hz rate; each burst consisted of 3 pulses delivered at 50 Hz rate. For intermittent TBS (iTBS), the bursts were applied for 2 s, with 8 s inter-burst-intervals, for a total of 600 pulses. For continuous TBS (cTBS), the bursts were applied continuously, also for a total of 600 pulses (Huang et al., 2005). For placebo stimulation a sham TBS was applied using the same stimulation parameters as with real iTBS, but using placebo coil (Magstim Co., UK), which looks the same as the real coil and, produces similar sound and local sensation as the real coil. All interventions were delivered over the non-dominant motor cortex. The muscle activity was carefully monitored by real-time EMG to confirm the relaxed status during the stimulation.

2. Materials and methods 2.5. Motor tasks 2.1. Subjects A total of 30 right handed (Oldfield, 1971) healthy people (18 men) aged 26 ± 3 years participated in this study. They were not taking any medication, and had no relevant medical history. The

Two motor tasks were used for the assessment of motor learning – a simple one, the simple reaction time task (RTT), and a complex one, Purdue pegboard task (PPT). The RTT consisted of a rapid squeezing of a rubber oval object with thumb and index

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finger in response to a buzzer. Ten trials were carried out at each of the assessment time-points. The inter-trial interval randomly varied between 6 and 10 s. The variable measured was time between the sound stimulus and the beginning of the FDI contraction in the EMG. The measured values were averaged for each of the assessment points separately. Only the target non-dominant hand was tested. The PPT is a much more complex motor task that requires both, manual dexterity and visual-motor coordination (Strauss et al., 2006). As such, it is comparable with real-life manual skills that people are required to learn throughout their lifetime. Given the increasing interest for using non-invasive brain stimulation in neurorehabilitation, studying the effects of TBS on a realistic model of motor skill learning could have stronger clinical relevance than the results obtained with simple ballistic movements that have been used in previous studies. The PPT was carried out using so-called Purdue pegboard (Lafayette Instruments model 32020, Lafayette, IN, USA). This is a rectangular wooden board with two longitudinally oriented parallel rows of holes, along the board’s longer axis, and four shallow wells at the top of the board arranged in a line perpendicular to the holes’ lines; two of the wells (the leftmost and the rightmost ones) contain small metal pegs (Fig. 1). The task used in the study required from the participants to pick individual pegs, using their thumb and index finger, and to place them into the holes in the pegboard as fast as they can. The pegs were picked from the well and then placed in the row of holes on the side closer to the hand performing the task. During the task, the participants were seated in a chair with the tested arm resting comfortably on the pegboard that was firmly clamped to a table. The height of the chair and position of the pegboard were adjusted to allow unrestricted, comfortable movement of the arm over the entire surface of the pegboard. The single PPT trial lasted 30 s. The participants were encouraged to place as many pegs as possible and the number of pegs placed on each trial was recorded. At each assessment time-point, the participants performed three trials with each hand, dominant (D-PPT) and non-dominant (ND-PPT); the inter-trial interval varied between 20 and 30 s. The mean of the three trials was taken as a measure for further analyses. 2.6. Experimental design The experimental design is shown in the Fig. 2. At the baseline (T0), before an intervention, PPT, RTT, RMT, AMT, and MEP were measured. Subsequently, a TBS intervention was applied.

Immediately after the intervention (T1), and 30 min later (T2), the MEP, RTT, and PPT were measured. The break between the two series of measurements was approximately 15 min. As it can be seen from the Fig. 2, at time T0 the PPT was first performed by a dominant and then a non-dominant hand (D-PPT and ND-PPT, respectively), whereas after the stimulation the order was reversed. This was done to prevent possible transfer of learning from the dominant hand to the non-dominant hand (Kumar and Mandal, 2005) in post-intervention measurements. That is, we wanted to assess the performance of the target non-dominant hand just before and immediately after the intervention, without any interference from the dominant hand, to get an un-obscured evaluation of the intervention effect. 2.7. Statistical analyses At the baseline, comparisons between groups were carried out by one-way analysis of variance (ANOVA) with factor Group, using absolute values, except for gender distribution where Chi-Square Test was used. Further analyses were conducted on normalized data; the normalization was carried out using baseline (T0) values, using the formula: nTx = 100  (Tx T0)/T0, where Tx stands for the absolute value at a time point, T0 stands for the absolute value at baseline, and nTx stands for normalized value at the time point. To check for the isolated effect of task repetition on performance, i.e. spontaneous learning effect, repeated measures one-way ANOVAs were carried out on RTT and PPT results from the placebo group, with time (T0 vs. T1 vs. T2) as repeated factor. The groups did not differ in their RTT results at baseline, thus between-group differences (i.e. the interventions’ effects) for RTT were assessed by a mixed-design two-way ANOVA with non-repeating factor Group (iTBS, cTBS and placebo) and repeating factor Time. However, the baseline PPT achievements varied among groups and therefore the intervention-related between-group differences for PPT were assessed using the mixed-design two-way ANOVA with analysis of covariance (ANCOVA), with same factors as for RTT, but with the baseline PPT as covariate. The least-square difference (LSD) test was used for post hoc pair-wise analyses. Correlations between post-interventions’ changes in cortical excitability (i.e. MEP sizes) and changes in tasks’ performance were tested separately for each of the 2 post-interventional segments, T0–T1 and T1–T2, by calculating Pearson correlation coefficients. The threshold for significance for all analyses was set at P < 0.05. 3. Results

Fig. 1. The Purdue pegboard is a rectangular wooden board with two longitudinally oriented parallel rows of holes, along the board’s longer axis, and four shallow wells at the top of the board arranged in a line perpendicular to the holes’ lines; two of the wells (the leftmost and the rightmost ones) contain small metal pegs. The participants have to pick individual pegs from a well, using their thumb and index finger, and to place them into the holes in the pegboard as fast as they can. The pegs were picked from the well and the pegs were placed in the row of holes on the side closer to the hand performing the task.

None of the participants reported any unpleasant sensation during the delivery of TBS, nor did they report any side-effects afterwards (e.g., headaches, drowsiness). At the baseline (T0), there was no difference between groups regarding the mean age of the participants, gender distribution, the lateralization quotient, RMT, and the mean MEP size (p > 0.66 in all cases). Placebo group had mean baseline RTT of 0.21 ± 0.03 s. which was similar with the RTTs measured in iTBS (0.20 ± 0.01 s.) and cTBS (0.21 ± 0.02 s.) groups (F(2,27) = 0.495, p = 0.615). However, three groups, placebo, iTBS, and cTBS differed slightly in baseline PPT values. One-way ANOVAs showed significant differences between groups for both, ND-PPT (16 ± 1.4, 15 ± 1.3, and 17 ± 1.7, respectively; F(2,27) = 5.639, p = 0.009) and D-PPT (17 ± 1.3, 16 ± 1.3, and 18 ± 1.4, respectively; F(2,27) = 4.603, p = 0.019). However, post hoc analyses showed that the only difference was between the cTBS and the iTBS groups (ND-PPT: p = 0.002; D-PPT: p = 0.006), while neither differed from the placebo group (for all comparisons: p > 0.05).

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Fig. 2. The experimental design. PPT – Purdue pegboard test; RTT – reaction time; RMT – rest motor threshold; AMT – active motor threshold; MEP – motor evoked potentials. D–ND – dominant hand was tested first; ND–D – non-dominant hand was tested first.

3.1. Excitability A two-way ANOVA (Table 1) showed as significant effects of the factor Group and of the Time  Group interaction. Post-hoc analyses showed large differences between groups (Fig. 3a). The placebo group experienced no changes in excitability (for all comparisons p > 0.528). In the iTBS group, the excitability increased at T1 (T0 vs. T1: p = 0.000), but subsequently dropped down (T1 vs. T2: p = 0.002), and after 30 min it returned to the initial baseline level (T0 vs. T2: p = 0.194). In cTBS group, the level of excitability decreased immediately after stimulation (T0 vs. T1: p = 0.000) and did not change afterwards (T1 vs. T2: p = 0.264), remaining below the baseline level 30 min after the intervention (T0 vs. T2: p = 0.000). The different within-group patterns of post-interventional changes in excitability were reflected in the between-group differences at various time-points as well (Fig. 3a). Immediately after stimulation (T1) both experimental groups differed from the placebo group (iTBS vs. placebo: p = 0.002; cTBS vs. placebo: p = 0.001). However, 30 min after the stimulation (T2), only the cTBS group still had excitability different to the placebo group (cTBS vs. placebo: p = 0.002), while excitability in the iTBS group returned toward the placebo group values (iTBS vs. placebo: p = 0.628). 3.2. Motor tasks 3.2.1. Spontaneous learning in placebo group The RTT values were similar at all time-points (Fig. 3b) and did not vary significantly (F(2) = 0.132, p = 0.878). In contrast, PPT values progressively increased with task repetitions for both, Table 1 ANOVA1/ANCOVA2 results on experimental measures. For D-PPT and ND-PPT, covariate was corresponding absolute PPT result at baseline (B). Time (F(df, error MEP1 RTT1 D-PPT2 ND-PPT

2

0.812 (2, 26) 0.865 (2, 26) 0.284 (2, 25) 0.770 (2, 25)

df))

Time  Group (F(df, error df))

Group (F(df, error

10.552** (4, 52) 0.179 (4, 52) 0.662 (4, 52) 3.358* (4, 52)

21.112** (2, 27) 0.273 (2, 27) 0.085 (2, 26) 3.829* (2, 26)

df))

Covariate (F(df, error df)) – – 0.024 (1, 26) 0.309 (1, 26)

MEP – motor evoked potential size; RTT – reaction time test; D-PPT – normalized results on PPT with dominant hand; ND-PPT – normalized results on PPT with nondominant (target) hand. * p < 0.05. ** p < 0.001.

non-dominant and dominant hand (Fig. 3c and d) (F(2) = 13.076, p = 0.003; F(2) = 28.869, p = 0.000; respectively). Post-hoc pair-wise analyses showed similar pattern of change for both hands, modest increase in T1 (T0 vs. T1: p = 0.047 and p = 0.013, respectively) and much larger increase in T2 (T1 vs. T2: p = 0.003 and p = 0.002, respectively). 3.2.2. The effects of the interventions The RTT values were similar in all tested groups across all time-points (Fig. 3b). ANOVA showed no significant effect either of factor Group or of Time  Group interaction (Table 1). For ND-PPT (Fig. 3c), ANCOVA showed significant effects of factor Group as well as of Time  Group interaction, while there was no significant effect of the covariate, the PPT at baseline (Table 1). The post hoc analyses showed different pattern of motor learning between groups. Within groups’ comparisons showed that while the participants in the placebo group were learning continuously, albeit quite modestly, from T0 to T1 (p = 0.047) and much more from T1 to T2 (p = 0.000), the participants in the iTBS group learned markedly immediately after the intervention (T0 vs. T1: p = 0.000), but not afterwards (T1 vs. T2: p = 0.513), although they kept their achieved level of improved performance (T0 vs. T2: p = 0.000). In contrast to them, the participants in the cTBS group did not learn immediately after the intervention (T0 vs. T1: p = 0.180), but did learn quite considerably afterwards (T1 vs. T2: p = 0.000), achieving levels of performance well above the baseline (T0 vs. T2: p = 0.001). Between-groups’ analyses showed that the participants from the cTBS group learned significantly less than participants from the placebo group at both T1 and T2 (p = 0.021, and p = 0.029, respectively). In contrast, there was no significant difference between participants from the iTBS and placebo groups at either T1 or T2 (p = 0.104, and p = 0.275, respectively). For D-PPT (Fig. 3d), ANCOVA did not show a significant effect of the factor Group or of the Time  Group interaction; there was no significant effect of the covariate, the PPT at baseline, either (Table 1). The participants from all groups showed similar improvement in learning over time (for all groups together, post hoc analyses: T0 vs. T1, p = 0.000; T1 vs. T2, p = 0.003). 3.3. Correlations Correlation coefficients between changes in MEP size and changes in RTT were not significant for either of the time intervals, T0–T1 and T1–T2. In contrast, correlation coefficients between changes in MEP size and changes in ND-PPT (Fig. 4) were significant for time interval T0–T1, but not for the time interval T1–T2 (r = 0.502, p = 0.005; and r = 0.073, p = 0.70; respectively).

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Fig. 3. Results for all three groups presented together. (a) Motor evoked potentials (MEP). (b) Reaction time (RTT). (c) Purdue pegboard test (PPT) with the non-dominant (target) hand (PPT-ND). (d) PPT with the dominant hand (PPT-D). All data are presented as relative change from the baseline (T0). Significant differences detected by pair-wise post hoc tests are marked by different symbols: ⁄ – comparison vs. T0, # – comparison vs. placebo, & – comparison vs. T1. Only analyses with significant Time  Group interaction in ANCOVA are marked.

a

b

Fig. 4. Correlations between relative changes in MEP size (DMEP) and changes in ND-PPT (DPPT) analyzed separately between T0 and T1 (a), and T1 and T2 (b). For each timepoint pair the data are presented as relative to the earlier time-point; i.e. for T0–T1 comparisons formula used was 100  (T1 T0)/T0, while for T1–T2 it was 100  (T2 T1)/ T1.

4. Discussion 4.1. Excitability The principal aim of this study was to compare directly the effects of the two TBS types and to check whether changes in M1 excitability induced by TBS would affect early motor learning. As it was expected from published data, following the iTBS the M1 excitability increased (Huang et al., 2005, 2007, 2008; Di Lazzaro

et al., 2011), while following the cTBS the M1 excitability was reduced (Huang et al., 2005, 2007; Iezzi et al., 2010); application of the sham TBS (placebo group) did not cause any change in the M1 excitability (Agostino et al., 2008; Iezzi et al., 2010; Jelic et al., 2013). It is of note that M1 excitability change lasted much longer following cTBS than following iTBS. The reduced excitability following cTBS lasted unchanged for 30 min following the intervention, which was in keeping with initial report by Huang et al.

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(2005) that cTBS after-effects could be seen as long as 60 min following the intervention. In contrast, the M1 excitability increase following iTBS did not last as long and was already absent at 30 min following the intervention (i.e. at T2 time-point). A number of other studies reported similar results (Huang et al., 2005; Gamboa et al., 2010; Di Lazzaro et al., 2011; Cardenas-Morales et al., 2014). Moreover, the very nature of the behavioral function investigated in this study required from participants to execute various motor tasks before and following the intervention. Motor activity was shown to be able to alter iTBS effects on M1 excitability (Huang et al., 2008; Iezzi et al., 2008) and that also might be the reason for the findings in this study. It may be of relevance that in studies which reported longer-lasting post iTBS M1 excitability increase participants were at rest during the post interventional follow-up period (e.g. Suppa et al., 2008). 4.2. Motor tasks On that backdrop, application of TBS protocols over non-dominant right M1 induced distinctive changes in the performance with the contralateral (i.e. target) hand. In comparison to the placebo, inhibitory cTBS protocol induced an overall slowing of the motor learning, while facilitatory iTBS protocol, although not inducing speeding up of the motor learning, as might have been expected, induced a change in the motor learning pattern. 4.2.1. TBS and reaction time test Reaction time task used in this study was a rather simple motor task, the aim of which was to test elementary motor performance, mainly the motor speed. The performance on that task has not changed with repetition in the placebo group signifying a lack of learning effect. Likewise, neither of the TBS protocols appeared to have any influence, despite the induced changes in M1 excitability. Similarly, Ragert et al. (2009) found that following excitatory iTBS over non-dominant M1, that was applied subsequently to a placebo rTMS over the dominant M1, there was no change in the RT despite increase of the M1 excitability of the non-dominant M1. The RTT requires relatively short time to be performed (200 ms) and thus its execution most likely does not require particularly strong activation of motor cortical areas. The physiological level of activation of the M1 required for optimal performance of the RT in healthy people is obviously rather low in comparison to the available functional reserve. Therefore, externally induced changes in M1excitability are not able to cause a significant change in performance. In keeping with this, internally increasing M1 excitability by voluntary tonic pre-contraction of the responding muscle seems not to change performance on RTT as well (Riedo and Ruegg, 1988). It has to be noted that Huang et al. (2005) reported changes in RT induced by cTBS. However, they used a variant of choice reaction time task where participants had to decide with which hand to respond. Their task involved not only simple stimulus–response loop, as the task used in our study, but required involvement of interhemispheric cooperation and decision making processes and was thus cognitively much more complex than RT task in our study. In general, tasks that are cognitively more complex might be more sensitive to the effects TBS and other neuromodulatory interventions than tasks with little or no cognitive involvement. In this study, this was apparent in results obtained with PPT. It is also of note that studies on post-stroke patients (Talelli et al., 2007; Sung et al., 2013) reported different results with RT than the results we had with healthy participants. Increase of M1 excitability of the affected hemisphere by TMS interventional methods was associated with significant shortening of the affected hand’s RT. It can be assumed that following stroke the spontaneous activity of affected M1 and functionally related structures during

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RT task execution is below the level required for optimal performance thus allowing for externally induced increase of M1 excitability and activation to bring about improvements in RT performance. The mechanisms behind the apparent failure of spontaneous M1 activation to achieve the level required for optimal performance would need to be clarified. 4.2.2. TBS and Purdue pegboard test In contrast to RTT, performance on the more complex PPT improved significantly with repetition, not only in the placebo group but also in the both TBS groups, signifying clear learning effect (Schmidt and Lee, 2005). The results were in keeping with previous results with PPT (Reddon et al., 1988; Noguchi et al., 2006; Weiler et al., 2008). 4.2.2.1. Performance of the ipsilateral (non-stimulated) hand. Course of motor learning on the PPT with dominant hand (ipsilateral to the site of stimulation) was similar for all three groups. Neither of the two TBS methods had any obvious effect on learning with ipsilateral hand. This may be due to the rather selective activation of only limited sub-populations of M1 interneurons by TBS (Huang and Rothwell, 2004), which may not have strong projections towards contralateral hemisphere. In keeping with this, (Di Lazzaro et al., 2010, 2011) did not find any change in excitability of the opposite hemisphere following TBS protocols. Similarly, Stefan et al. (2000) did not find any change in contralateral M1 excitability following cTBS applied over non-dominant M1. The lack of excitability change in the opposite hemisphere following TBS protocols could be a reason for absence of difference between learning curves in placebo group and the two TBS groups. 4.2.2.2. Performance of the contralateral (stimulated) hand. In contrast to the ipsilateral hand, the course of motor learning with contralateral non-dominant hand differed between groups (Fig. 2c). The two TBS protocols, besides inducing opposite changes in M1 excitability, induced alteration in the PPT learning curves that made them deviate from the learning curve in placebo group in distinctively opposite directions. In this study, the interventional TBS protocols were administered during period of rest following the first block of the task performance, the time window during which a process of early consolidation of learning would be supposed to take place (Brashers-Krug et al., 1996; Shadmehr and Holcomb, 1997; Krakauer and Shadmehr, 2006; Tanaka et al., 2011). Therefore, it could be assumed that the main impact of the TBS interventions was on that phase of the learning process. Unfortunately, the baseline difference in PPT achievements between iTBS and cTBS groups’ participants has made direct comparisons between the two TBS interventions less certain. The iTBS group’s inferior baseline performance might have left them more space for improvement with further practicing then it was the case for cTBS group which was superior from the onset. Alternatively, the iTBS group’s low baseline PPT scores may also signify their lower overall dexterity, which might be also linked with their lower capacity for improvement with practice. If the former were true, differences between effects of the two TBS interventions would be spuriously exaggerated, while if the later were true, the differences would be falsely reduced. Therefore, since baseline PPT scores from neither of the TBS groups differed from the scores of the placebo group, we limited our discussion to differences between placebo and each of the two TBS interventions separately. Following the iTBS, which caused increase of M1 excitability immediately after the intervention with return to the baseline level in the later course, the PPT learning curve showed a sharp rise immediately after the intervention, but afterwards remained more-or-less steady, maintaining the achieved level of improved performance. This pattern was in clear contrast with the pattern

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seen following placebo intervention where learning curve showed more gradual but continuous rise, which was even considerably larger in the later course than immediately after the intervention. However, despite these differences, the ultimate progress in learning of the motor task was similar for both groups and no significant difference was found between the groups. Similar lack of significant increase of learning achievements in spite of the significant increase of M1 excitability following M1 iTBS was found by Agostino et al. (2008). They investigated the effects of left hemisphere M1 iTBS on learning of the fast abduction movements with right index finger. The intervention was applied during consolidation, between the blocks of task performance, but no difference was found between participants who received iTBS and those who received placebo. In contrast, using similar motor task (ballistic thumb abductions), Teo et al. (2011) found significant improvement of motor learning with non-dominant hand following right hemisphere M1 iTBS in comparison to placebo stimulation. However, they applied iTBS before the performance of the motor task had started, i.e. before the learning process had started, which differed from Agostino et al. (2008) and this study where iTBS was applied during the early learning consolidation phase. It may be that iTBS induced increase of M1 excitability has different effects depending on the phase of the learning process during which it occurs. Immediately before motor learning, it can improve the subsequent learning process, while if it is timed during the learning process it may change the pattern of the learning curve but will not change the overall learning effects. The effects of the cTBS were considerably more striking than iTBS effects. The cTBS induced reduction of the M1 excitability was associated with significantly worse learning in comparison to placebo group, both immediately after the intervention and 30 min later. Instead of gradual but significant increase of the learning curve as seen following placebo, following cTBS learning curve remained initially flat only to start rising later on. The overall learning achievement was thus well below the one seen following placebo. Consistent with these findings are the results of Muellbacher et al. (2002) and Baraduc et al. (2004) studies, where the decrease in the M1 excitability was caused by another TMS protocol (1 Hz rTMS). The intervention in those studies was also applied during consolidation phase, i.e. between the series of fast ballistic movements (pinching movement in Muellbacher et al. (2002) study, fast index finger abduction in Baraduc et al. (2004) study). In both studies, the intervention significantly slowed motor learning in comparison to placebo. Moreover, Iezzi et al. (2010), using fast index finger abduction task with dominant hand, but applying cTBS immediately before motor learning, also had similar results. It seems as an externally induced reduction of M1 excitability, regardless whether occurring before start of the motor learning process or during it, can significantly impair early phases of a skill acquisition. Although the results of this study suggest quite strongly that TBS can induce changes in PPT performance, the precise mechanism behind this is not clear yet. The TBS may affect gross movements of the forearm changing thus the outcome of PPT in a rather unspecific way. However, this does not seem very likely since the TBS was delivered focally and targeted towards cortical representations of the small hand muscles. Alternatively, it may be simply influencing the speed of finger movements. This also is not very likely given the lack of any TBS effect on RT where essentially only the speed of finger movements was assessed. We believe that observed TBS effects on motor learning most likely have taken effect through modulation of synaptic connections between subsets of M1 interneurons responsible for precise coordination of muscle synergies and co-activations during task execution.

4.3. M1 excitability – motor learning correlations Comparison of the ND-PPT learning curves (Fig. 3c) with the M1 excitability change curves (Fig. 3a) suggests some interesting possible associations. Externally induced increases and reductions of M1 excitability were associated with corresponding changes in learning of the PPT performance. Increase of excitability, as it could be seen during T0–T1 interval following iTBS, was associated with a trend towards better PPT learning than it did happen in placebo group. However, even more impressive was association between reduction of excitability and halting of the learning process, as it could be seen not only during T0–T1 interval following cTBS, but also during T1–T2 interval following iTBS. In contrast, maintenance of the M1 excitability at the more-or-less unchanged level, regardless of whether below, above or at the baseline level, coincided with restitution of the learning curve slope that was similar with the slope of the learning curve following placebo at the corresponding interval. This was obvious during T1–T2 interval where learning curves following cTBS and placebo were almost parallel. The described associations were partially confirmed by the results of the correlation analyses which showed significant positive correlation between excitability change and PPT performance change (i.e. learning curve slope) during T0–T1 interval for all participants together. It seems as the TBS induced promotion/suppression of the M1 functional plasticity reserves, represented by the magnitude of change in excitability, has much greater influence on early consolidation of the learned material than the absolute level of the M1 activation, represented by the sheer level of excitability. The obtained results also suggest that cTBS induced decrease in M1 excitability is able to interfere effectively with learning processes. In contrast, iTBS induced increase in M1 excitability does not seem to be particularly potent tool for inducing improvements in motor learning, at least when applied in between the training sessions. However, the situation may be different if the intervention is applied before any training takes place. In addition, it would be interesting to check whether subjects’ performance could be reinforced by applying iTBS after the learning protocol had took place, during the period of late consolidation. The results of this study may help in better defining the use of TBS for the promotion of motor learning in neurorehabilitation (Thickbroom and Mastaglia, 2009) and cognitive enhancement (McKinley et al., 2012). However, given the high inter-individual variability of TBS effects on M1 excitability (Hamada et al., 2013) it is also necessary to better examine the degree of inter-individual variability of TBS effects on motor learning processes in larger population of participants. Acknowledgments Authors would like to thank Professor John Rothwell for his helpful comments and advices, and Mr. Vuk Stevanovic´ for his help in data acquisition. This study was supported by project grant (#175012) from the Ministry for Education, Science and Technological Development of Republic of Serbia. Authors have nothing else to disclose. Conflict of interest: There has been no conflict of interest References Agostino R, Iezzi E, Dinapoli L, Suppa A, Conte A, Berardelli A. Effects of intermittent theta-burst stimulation on practice-related changes in fast finger movements in healthy subjects. Eur J Neurosci 2008;28:822–8. Baraduc P, Lang N, Rothwell JC, Wolpert DM. Consolidation of dynamic motor learning is not disrupted by rTMS of primary motor cortex. Curr Biol 2004;14:252–6. Brashers-Krug T, Shadmehr R, Bizzi E. Consolidation in human motor memory. Nature 1996;382:252–5.

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