Supplementary figure: Kantak, Sullivan, Fisher, Knowlton and Winstein

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1 Supplementary figure: Kantak, Sullivan, Fisher, Knowlton and Winstein Supplementary figure1 : Motor task and feedback display during practice (A) Participants practiced an arm movement task aimed to match a target presented on the screen. There were 4 targets: A1, A2, A3 and A4, each with a different absolute amplitude specifications (30, 45, 60 and 75 degrees, respectively), but a similar movement structure and same absolute time requirements (800ms). The constant practice groups practiced 120 trials of target A3, while the variable practice group practiced 60 trials of target A3 and 20 trials of targets A1, A2 and A4 each. The order of presentation of the four targets was randomized in the variable practice groups. (B) Example of the feedback display. Participant movement trajectory (thin line) superimposed on the target trajectory (thick line). The root mean square error (RMSE) was displayed along with the trajectories after each trial.

2 SUPPLEMENTARY METHODS: Material and Methods: Participants: Fifty nine right-handed volunteers (25 men, 34 women; mean age years; range: years), naïve to the purpose of the experiments, participated. All participants were free of any history of neurological disorders, and met the safety criteria for repetitive Transcranial Magnetic Stimulation (rtms) and Magnetic Resonance Imaging (MRI). A written informed consent, approved by the Institutional Review board of the University of Southern California was obtained prior to participation. The participants were randomized in to six experimental groups resulting from three rtms sites (none, M1, DLPFC) by two practice structures (Constant, Variable): Constant practice no-rtms (CP, n=10), Variable practice no-rtms (VP, n=11), Constant practice M1 Interference (CP-M1, n=10), Variable practice M1 interference (VP-M1, n=10), Constant practice DLPFC interference (CP-DLP, n=9) and Variable practice DLPFC interference (VP-DLP, n=9) (Fig. 1). Motor task: Participants practiced moving a light weight lever with their dominant arm to replicate a target trajectory (a position-time trace) presented on a computer monitor. This lever was affixed to a frictionless vertical axle restricting movement of the lever to the horizontal plane above the surface of a table. The handle at the end of the lever was adjusted to accommodate the length of the participant s forearm. A linear potentiometer, attached to the base of the vertical axle recorded lever-position information with movements of the lever away from the body reflecting upward movements on the computer monitor and movements toward the body reflecting downward movements on the computer monitor. Signals from the potentiometer were converted to digital signal by an A/D board of a Compact 466v computer and sampled at 1000Hz to provide feedback (FB) on the computer monitor. A custom template software program (A.Weekly, 2004) was used for manipulation of the movement trajectory and the interval duration, and data storage for off-line analysis of each trial. There were 4 target trajectories (position-time traces): A1, A2, A3 and A4 (Supplementary Figure A), each having different peak amplitude specifications (30, 45, 1

3 60 and 75 degrees) respectively, but with a similar movement structure and temporal duration requirements (800ms). The coordinated arm movement skill was to replicate a target trajectory that consisted of two elbow extension-flexion reversal movements, each of specific amplitude, performed in the horizontal plane. This was a fast movement with a total duration of 800 ms. One of the 4 target trajectories was displayed on the computer monitor at the beginning of each trial for a 2000 ms duration, after which the trajectory disappeared from the screen. After 1000 ms, a go signal was displayed at which point, the subject was instructed to move the lever in a manner to replicate the target trajectory as closely as possible. After a 2000 ms delay following movement, postresponse feedback was displayed on the computer screen for a 5000 ms duration. Feedback consisted of: 1) an overall numeric error score (root mean square error, RMSE) and 2) a graphic representation of the participant s response, time-locked to onset and superimposed on the target movement pattern (supplementary figure. B). Assessment of Motor corticospinal excitability: On day 1, prior to randomization, motor corticospinal excitability was assessed in all participants using TMS. Stimulation was delivered with a 70 mm figure of eight coil attached to Magstim Rapid2 magnetic stimulator. Single pulse TMS was used to identify the hot-spot which is the optimal scalp position for consistently eliciting the largest motor evoked potential (MEP) from the biceps brachii muscle. The coil was held tangentially to the scalp contralateral to the dominant arm with the coil-handle pointing posteriorly away from the midline at an angle of 45 1, 2. Next, resting motor threshold (MT) was determined by systematically decreasing the stimulus intensity over the hotspot. MT is defined as the lowest intensity level required to induce MEP peak-to-peak amplitude of at least 50 V, in 5 out of 10 consecutive trials 3. Baseline motor corticospinal excitability was assessed by applying 10 TMS pulses at 120%MT intensity over the hot-spot. Practice: Ten minutes following this procedure, each participant practiced the motor task for a total of 120 trials during the acquisition phase. The structure of the practice condition was different for the constant practice and variable practice groups. The constant practice groups (CP, CP-M1, and CP-DLP) practiced 120 trials of target A3 (criterion target). In the variable practice groups (VP, VP-M1, and VP-DLP), the participants practiced the target A3 for 60 trials and 20 additional trials of targets A1, A2 and A4 each. The presentation order of the four targets was pseudo-randomized in the 2

4 variable practice groups. During practice, participants sat comfortably in front of the computer monitor with their dominant forearm along the arm of the lever and their hand grasping the lever handle. A sample trajectory was used to orient the participant to the task. Sample goal movement and feedback were explained carefully to each participant. The experimenter and participants reviewed templates of a sample target trajectory and superimposed feedback trajectory to ensure maximal understanding of the computer displayed feedback. The participants were instructed that during practice, they were to practice to replicate the goal trajectory and make their movements as accurate as possible (i.e. lower RMSE). When the experimenter determined that the participant was adequately oriented to the task and feedback, practice was begun. Immediately following practice, each participant was tested for the end of acquisition performance (EoA). One day later (24 +3 hours), the participants returned for a retention test (R). It was essential that we test the effect of rtms on consolidation processes after the time-dependent effects of rtms have faded away. In this study we used a retention test after 24-hour to study the effects on memory. The EoA and 1 day retention test (R) consisted of a nofeedback, 4-trial block of the criterion target trajectory (A3). The M1 interference groups (CP-M1 and VP-M1) received 1 Hz rtms over the biceps representation of M1 after EoA (Fig. 1). The DLPFC interference groups (CP-DLP and VP-DLP) received 1Hz rtms over the DLPFC 4 similarly, immediately after EoA (Fig. 1). rtms procedure: 1 Hz rtms was applied with Magstim 70 mm figure of eight coil attached to a Magstim Rapid² magnetic stimulator. Immediately after EoA, subjects in the CP-M1 and VP-M1 groups received a 1 Hz train of rtms for 10 min (600 pulses) at 110% MT intensity over the hotspot of biceps brachii in contralateral M1. Immediately before and after rtms over M1, we measured MEP amplitude to examine rtms-induced changes in motor corticospinal excitability. In the CP-DLP and VP-DLP groups, 1Hz rtms at 110% MT intensity for 10 min (600 pulses) was applied over the contralateral DLPFC. A structural MRI-based, stereotaxic neuronavigation system, Brainsight Frameless (Rogue Research), was used to precisely localize the TMS coil over the DLPFC. First, a three dimensional (3D) image of the cerebral cortex was reconstructed in Brainsight by processing a two-dimensional MR image of each participant. This 3D image allowed identification of the sulci and gyri on the individual brain surface. Markers were placed on the MR image at specific anatomical landmarks: the tip and base of nose, right and left tragus of the ear. Then optical markers were attached to the TMS coil 3

5 which was calibrated and registered to the Brainsight system by means of a Polaris optical position sensor and a coil-tracker device. Next, the participant put on tracker glasses with reflective markers. The participant s anatomical landmarks were then coregistered with the anatomical landmarks on the MR image. Coregistration entails an optimal transformation between actual skin points on the participant and the skin surface of the MRI reconstructed human model. This allowed a real time display of the relative positions of the coil and the participant s head and brain surface, which was critical in guiding the placement of the coil over the DLPFC. Stimulation over DLPFC was directed at the middle third of the middle frontal gyrus corresponding with the region of Broadmann area 46, and posterior part of area 9 5. Data Analysis: Motor behavior: Performance accuracy was assessed for practice, end of acquisition (EoA) and retention test (R). Dependent measure for accuracy included RMSE which is the average difference between the goal movement trajectory and the participant s response, calculated over the participant s total movement time 6. RMSE was calculated for each trial. For the acquisition phase, only the trials on criterion task (A3) from the variable practice group, and corresponding trials in the constant practice group were included in behavioral analysis. Thus for each participant, we included the performance on the sixty trials of A3 for the acquisition phase analysis. For each participant, RMSE for these sixty trials were averaged into twelve 5-trial blocks for the acquisition phase. For the EoA and 1 Day retention test, an average RMSE was computed over each set of 4 criterion task trials. For motor skill practice data during the acquisition phase, a 2 practice condition (constant, variable) X 3 rtms site (no rtms, M1-rTMS and DLP-rTMS) X 12 Block (1-12 acquisition blocks) ANOVA with repeated measures on the last factor was used. Separate 2 practice condition (constant, variable) X 3 rtms site (no rtms, M1-rTMS and DLP-rTMS) ANOVA was used for EoA. To test for contextual interference effect with the present motor task, we compared the no-rtms groups (VP and CP) using a 2 practice condition (constant, variable) X 2 test (EoA, R) ANOVA with repeated measures on the last factor. 2 practice condition (constant, variable) X 3 rtms site (no rtms, M1-rTMS and DLP-rTMS) X tests (EoA, R) repeated measures (RM) ANOVA with RM on tests was used to compare the groups for offline performance changes from EoA to R. Separate 2 rtms site X 2 tests (EoA, R) RM 4

6 ANOVA was conducted to compare the rtms interference groups to the control groups. The rtms-4 hr groups (CP-M1-4hr and VP-DLP-4hr) and rtms groups (CP-M1 and VP- DLP) were compared using a 2 (rtms, rtms 4h) X 2 test (EoA, R) RM ANOVA for each practice structure. For all statistical tests, the significance level was set at P<.05. SPSS version 15.0 statistical software was used for all statistical analyses. TMS data: MEPs were analyzed offline with a customized MATLAB (Mathworks, MA, USA) software tool DataWizard (version 0.8.5, Dr. Allan D. Wu, UCLA). Peak-to-peak amplitude was computed for each recorded MEP. For the M1-interference groups (CP- M1, VP-M1), mean MEP amplitude of 10 trials was calculated for baseline, pre- and post-rtms. A 2 practice(constant, variable) X 3 time points (baseline, pre- and post rtms) Repeated measures ANOVA with repeated measures on time points was compare the effects of practice and rtms on motor corticospinal excitability. A paired sample t test was used to compare the change in the motor cortical excitability following rtms compared to pre-rtms in the entire M1-interference group. 5

7 References 1. Brasil-Neto, J.P., McShane, L.M., Fuhr, P., Hallett, M. & Cohen, L.G. Topographic mapping of the human motor cortex with magnetic stimulation: factors affecting accuracy and reproducibility. Electroencephalogr Clin Neurophysiol 85, 9-16 (1992). 2. Mills, K.R., Boniface, S.J. & Schubert, M. Magnetic brain stimulation with a double coil: the importance of coil orientation. Electroencephalogr Clin Neurophysiol 85, (1992). 3. Maeda, F., Keenan, J.P., Tormos, J.M., Topka, H. & Pascual-Leone, A. Modulation of corticospinal excitability by repetitive transcranial magnetic stimulation. Clin Neurophysiol 111, (2000). 4. Muellbacher, W., et al. Early consolidation in human primary motor cortex. Nature 415, (2002). 5. Farzan, F., et al. Suppression of gamma-oscillations in the dorsolateral prefrontal cortex following long interval cortical inhibition: a TMS-EEG study. Neuropsychopharmacology 34, (2009). 6. Schmidt, R.A. & Lee, T.D. Motor control and learning: A behavioral emphasis (Human Kinetics, 2004). 6

8 Supplementary results: Kantak, Sullivan, Fisher, Knowlton and Winstein SUPPLEMENTARY RESULTS: Motor skill performance during practice and End of Acquisition (EoA) Participants in all groups improved across the acquisition phase as evidenced by a reduction in the global error (root mean square error, RMSE) in performance of the target matching task (practice effect; ANOVA, F 1, 58 = , p< 0.001). However, there was no significant difference between the groups across the acquisition phase (ANOVA, F 2, 53 = 0.458, p=0.635). At the first practice block, there was no significant difference in the skill performance on the criterion task (A3) between the six experimental groups (2X3 ANOVA; F (5, 58) = 1.324; p= 0.268). Additionally, end of acquisition (EoA) performance on the no-feedback test was not significantly different between the experimental groups (ANOVA, F 2, 58 = 0.463, p=0.632). Motor corticospinal excitability: We assessed the effect of practice conditions and 1Hz rtms over M1 on motor corticospinal excitability by examining the change in the MEP amplitude at three time points: baseline, pre- and post- rtms in CP-M1 and VP-M1 groups. There was no significant difference between the two groups at the three time points (2X3 Repeated measures ANOVA, F 1, 18 = 0.152, p = 0.702). Further, 1Hz rtms at 110%MT applied over M1 for 10 min reduced motor corticospinal excitability as evidenced by a significant reduction in MEP amplitude post rtms (Mean MEP amplitude V + SEM: ) compared to pre-rtms (Mean MEP amplitude V + SEM: ) (Paired sample t test, t (19) = 4.377; p< 0.05). This finding confirmed that 1Hz rtms was a valid tool to down regulate motor corticospinal excitability and induced a virtual lesion that could be used to interfere with cortical processing during early motor memory consolidation 1

9 Supplementary discussion: Kantak, Sullivan, Fisher, Knowlton and Winstein SUPPLEMENTARY DISCUSSION: Animal and human imaging studies suggest that the DLPFC is involved in mechanisms of attention, processing of working memory 1, representation of goal-related spatial information 2, selection and planning of upcoming actions 3, 4,formation of long-term memory 5, 6, as well as consolidation 7. In our study, variable practice required the participants to attend to the task, select, and execute a different movement plan at each trial as prompted by the target display. This may require a relatively higher engagement of the DLPFC compared to that of M1. Constant practice required the learner to move repeatedly to only one target across practice. Therefore, unlike variable practice, the learner may not be forced to rely as much on working memory, action selection, and planning that garner the unique processing capabilities of DLPFC. Instead, repeated practice on a single task may result in memory formation within M1 and this may be a critical node for motor memory consolidation after constant practice. M1 is involved in formation and maintenance of motor memory. Animal and human studies demonstrate that M1 processing early consolidation is critical for retention of motor skills Previous investigations have demonstrated that the role of M1during motor memory consolidation depends on the motor task characteristics 12, 13. Here we extend our understanding of M1 function by demonstrating that its role during consolidation is also modulated by the structure of practice. M1 processing is critical during memory consolidation following constant practice, and this consolidation involving M1 contributes to skill retention. Our finding that rtms post-variable practice did not impair retention was counterintuitive, especially in the light of previous investigations that reported M1activation during variable practice that significantly correlated with postpractice behavioral gains 14. It is possible that M1 processing is critical for motor memory encoding during, but not for consolidation immediately after variable practice. Alternatively, it is likely that M1 is engaged during consolidation after variable practice; but its involvement is not necessary for retention. Our findings, taken together with previous studies, suggest a dynamic nature of motor memory processing mediated by M1 that may, in part, depend on practice structure. One important consideration is that effects of rtms are possibly not restricted to stimulated M1 and DLPFC. There is evidence for remote effects of rtms via neuronal networks 15, 16, and this possibility cannot be ruled out in our study. We suggest that M1 1

10 Supplementary discussion: Kantak, Sullivan, Fisher, Knowlton and Winstein and DLPFC are critical components of neural circuits that mediate motor memory consolidation, and their engagement is modulated by practice structure. Tanaka and colleagues demonstrated that Supplementary Motor Area (SMA) is differentially engaged following each practice schedule 17. Our findings extend that line of work, and suggest that the neural substrates critical for consolidation of motor memory differ depending on the practice structure. Another possibility is that the difference is practice structure may actually trigger different post-practice processes mediated by distinct neural mechanisms. For example, reconsolidation may occur immediately after variable practice, while consolidation may follow constant practice. Behavioral literature suggests that variable practice, due to interspersing of tasks, may force the learner to forget an action plan for an executed task because he/ she has to perform a new task on the next trial. So, when the initial task is presented again, it provides opportunity for retrieval or reconstruction of action plan during practice 18. These retrieval processes during variable practice may render the motor memory relatively unstable, and trigger reconsolidation post-practice. In contrast, consolidation processes may follow constant practice, and engage M1. Distinct molecular mechanisms have been demonstrated for consolidation and reconsolidation 19. Consolidation may follow constant practice and rely on M1, while reconsolidation may follow variable practice and rely on DLPFC for retention. 2

11 Supplementary discussion: Kantak, Sullivan, Fisher, Knowlton and Winstein References 1. Fuster, J.M. Executive frontal functions. Exp Brain Res 133, (2000). 2. Genovesio, A., Brasted, P.J. & Wise, S.P. Representation of future and previous spatial goals by separate neural populations in prefrontal cortex. J Neurosci 26, (2006). 3. Pochon, J.B., et al. The role of dorsolateral prefrontal cortex in the preparation of forthcoming actions: an fmri study. Cereb Cortex 11, (2001). 4. Rowe, J.B., Toni, I., Josephs, O., Frackowiak, R.S. & Passingham, R.E. The prefrontal cortex: response selection or maintenance within working memory? Science 288, (2000). 5. Blumenfeld, R.S. & Ranganath, C. Dorsolateral prefrontal cortex promotes longterm memory formation through its role in working memory organization. J Neurosci 26, (2006). 6. Blumenfeld, R.S. & Ranganath, C. Prefrontal cortex and long-term memory encoding: an integrative review of findings from neuropsychology and neuroimaging. Neuroscientist 13, (2007). 7. Shadmehr, R. & Holcomb, H.H. Neural correlates of motor memory consolidation. Science 277, (1997). 8. Luft, A.R., Buitrago, M.M., Kaelin-Lang, A., Dichgans, J. & Schulz, J.B. Protein synthesis inhibition blocks consolidation of an acrobatic motor skill. Learn Mem 11, (2004). 9. Luft, A.R., Buitrago, M.M., Ringer, T., Dichgans, J. & Schulz, J.B. Motor skill learning depends on protein synthesis in motor cortex after training. J Neurosci 24, (2004). 10. Muellbacher, W., et al. Early consolidation in human primary motor cortex. Nature 415, (2002). 11. Robertson, E.M., Press, D.Z. & Pascual-Leone, A. Off-line learning and the primary motor cortex. J Neurosci 25, (2005). 12. Baraduc, P., Lang, N., Rothwell, J.C. & Wolpert, D.M. Consolidation of dynamic motor learning is not disrupted by rtms of primary motor cortex. Curr Biol 14, (2004). 13. Shemmell, J., Riek, S., Tresilian, J.R. & Carson, R.G. The role of the primary motor cortex during skill acquisition on a two-degrees-of-freedom movement task. J Mot Behav 39, (2007). 14. Wymbs, N.F. & Grafton, S.T. Neural substrates of practice structure that support future off-line learning. J Neurophysiol 102, (2009). 15. Paus, T., et al. Dose-dependent reduction of cerebral blood flow during rapid-rate transcranial magnetic stimulation of the human sensorimotor cortex. J Neurophysiol 79, (1998). 16. Van Der Werf, Y.D. & Paus, T. The neural response to transcranial magnetic stimulation of the human motor cortex. I. Intracortical and cortico-cortical contributions. Exp Brain Res 175, (2006). 17. Tanaka, S., Honda, M., Hanakawa, T. & Cohen, L.G. Differential Contribution of the Supplementary Motor Area to Stabilization of a Procedural Motor Skill Acquired through Different Practice Schedules. Cereb Cortex (2009). 18. Lee TD, S.D. Contextual Interference. in Information processing perspectives (2003). 19. Lee, J.L., Everitt, B.J. & Thomas, K.L. Independent cellular processes for hippocampal memory consolidation and reconsolidation. Science 304, (2004). 3