Subjects and Experimental Design
Sixty healthy volunteers (25 of them females, 27.7 ± 7.0 years) who were randomly assigned to 4 different groups (n = 15 each) participated in Experiment 1, which was geared to determine what effects the disruption of SMA activity with 1-Hz rTMS versus sham stimulation immediately after practice had on recall on Day 2 with the 2 different practice schedules (blocked and random). The 4 groups were as follows: (A) sham stimulation shortly after blocked practice, (B) 1-Hz rTMS over the SMA shortly after blocked practice, (C) sham stimulation shortly after random practice, or (D) 1-Hz rTMS over the SMA shortly after random practice (). In Experiment 2, 63 additional subjects (20 of them females, 25.0 ± 4.6 years), who were divided into 5 groups, were later recruited to study what effects the disruption of SMA 6 h after practice had ended and the stimulation of other cortical regions also shortly after practice had ended had on recall on Day 2: (E) 1-Hz rTMS to a control position 2 cm posterior to Cz (international 10–20 system, n = 12), (G) to the left M1 (n = 13) shortly after blocked practice, (H) to the left M1 shortly after random practice (n = 13), (I) to the left dorsal premotor cortex (PMd) shortly after random practice (n = 13), and (F) 1-Hz rTMS to the SMA 6 h after blocked practice (n = 12) ().
Figure 1. Task and experimental design. (A) Healthy volunteers were divided into 9 groups. In the main experiment, 4 groups were evaluated in a 2 × 2 factorial design, defined by the type of practice (blocked/random) and rTMS (sham/SMA). Five more groups (more ...)
The purpose of Experiment 1 (groups A–D) was to explore what differential effects a virtual lesion disruption of SMA (Pascual-Leone et al. 2000
) immediately postpractice had on recall on Day 2 with the 2 practice schedules, blocked and random. Groups E–I (Experiment 2) were added to characterize the temporal and spatial specificity of the effects of SMA stimulation on stabilization with the 2 practice schedules. For random practice, the M1 (Muellbacher et al. 2002
; Baraduc et al. 2004
; Robertson et al. 2005
; Lin et al. 2008
) and PMd (Shadmehr and Holcomb 1997
; Cross et al. 2007
) were targeted because previous studies have suggested the possible involvement of these structures in the stabilization of motor skills. All subjects were right handed and had given written informed consent before the experiments. None of them had a history of psychiatric or neurological illness. The experiment was approved by the National Institute of Neurological Disorders and Stroke Ethics Committee and the local Ethics Committee of the National Institute for Neuroscience.
All subjects came to the laboratory on 2 subsequent days (Days 1 and 2). On Day 1, they practiced 3 motor sequences (6 training blocks of 12 sequences per block for a total of 72 sequences. Each sequence was practiced 24 times in a blocked- or a random-practice schedule (). In the blocked-practice groups, they completed all practice trials of each sequence before proceeding to the next. In the random-practice groups, subjects practiced the 3 sequences intermixed in random order. The recall test on Day 2 consisted of 12 trials of the previously learned sequences (4 trials per sequence), and the order of sequences was counterbalanced across the subjects ().
Sequential Visuomotor Task
We used a sequential-visuomotor task similar to that described by Shea and Morgan (1979)
in which the contextual-interference effect was best described. Subjects sat in an armchair and visual stimuli were presented on a computer screen using a script based on Presentation software (Neurobehavioral Systems, Albany, CA). They were initially instructed to focus on a central fixation point (3–5 s) and subsequently warned of the upcoming trial by the presentation of a ready (red cross) signal for 0.5–2 s. After the ready signal disappeared, a visual cue composed of a red, blue, or green central square surrounded by 4 gray squares was presented. Each visual cue color was associated with a particular order of 5 subsequent mouse movements that started and finished at the central square (see for an example of a sequence time course and for the order of mouse movements in the 3 learning sequences). The subjects’ instructions were to move the screen's cursor with the mouse and click on each of the 4 targets one at a time until all targets were completed. Subjects were asked to be as fast and accurate as possible. The visual cue color was visible until subjects started to move the cursor and the center square changed color to gray at the onset of mouse movement. For each single mouse movement, if the chosen target was correct for that particular sequence, the target disappeared from the screen and subjects were required to move the cursor to the next target and click onto it. If it was the incorrect target, it did not disappear and they were required to move the cursor toward the other peripheral targets until they hit the correct one. For example, the correct sequence for the red visual cue was right-up, left-up, right-down, left-down, and central square (). For all sequences, the last target was always the square at the center of the screen. The intertrial intervals ranged randomly between 3 and 5 s. The response time (RT), that is, the primary end point measure of the study, was defined as the time between the onset of the presentation of the visual cue and the mouse click onto the last target stimulus (central square). For each participant, the median RT of 12 sequences (4 for each of the 3 practiced types: red, blue, and green in ) was calculated for each practice block on Day 1 (e.g., the value representing RT in Block 1 in each individual was the median of the first 4 red, the first 4 blue, and the first 4 green practiced sequences; in Block 2, it was the median of the second 4 red, the second 4 blue, and the second 4 green sequences, etc.) and at recall time on Day 2. Group data were calculated as the mean ± standard error of the individual median values. This analysis was required to compare improvements in performance over the practice time across the 2 training types (random and blocked). We also evaluated performance in an untrained, nonsequential, simple visuomotor task as a control-motor task. The purpose was to determine if the hypothesized disruptive effects of rTMS were specific to the newly learned sequential skill or represented a less specific effect on motor function in general (see Supplementary material
Transcranial Magnetic Stimulation
The rTMS was delivered from a Magstim Rapid Stimulator (Magstim Company, Whitland, UK) through 80-mm figure-eight coils that allowed delivery of real or sham stimuli. At the beginning of each experiment, we determined the resting motor threshold (rMT) for the right first dorsal interosseous (FDI) muscle over the left M1. The rMT was defined as the lowest intensity of transcranial magnetic stimulation (TMS) output required to elicit the motor-evoked potentials (MEPs) of at least a 50-μV peak-to-peak amplitude in at least 5 of the 10 consecutive trials (Rossini et al. 1994
). The coil was placed tangential to the scalp with the junction region pointing backward and laterally at a 45° angle away from the midline (Di Lazzaro et al. 2004
). In Experiment 1, we evaluated what effects 1-Hz rTMS over the SMA (15 min at 115% rMT intensity) or the sham applied shortly after practice had on recall on Day 2. The same intensity and duration of rTMS, which was started within 10 min after practice had ended in all groups, was used in all the experiments (115% rMT intensity for the FDI). The 1-Hz rTMS results in decreased excitability of the underlying cortical areas (Chen et al. 1997
; Robertson et al. 2003
) and can successfully downregulate activity in the SMA (Tanaka et al. 2005
; Perez et al. 2007
). When applied over the M1, this stimulation protocol decreases motor cortical excitability and influences the motor stabilization of tasks practiced in a blocked schedule (Muellbacher et al. 2002
; Baraduc et al. 2004
). The site of stimulation for each cortical area was determined using previously described procedures (Matsunaga et al. 2005
; Perez et al. 2007
; see Supplementary material
The electromyographic (EMG) activity was recorded from surface electrodes positioned on the skin overlying the FDI and tibialis anterior muscles in a bipolar montage (interelectrode distance, 2 cm). The EMG signals were amplified, filtered (band-pass, 25 Hz to 1 kHz), sampled at 2 kHz, and stored on a personal computer for off-line analysis.
The median RT of 12 sequences (including the 3 practiced sequences) was calculated for each participant for each practice block on Day 1 and at the recall time on Day 2. Repeated measures analysis of variance (ANOVA) was implemented with “practice” (blocked vs. random) as a between-subjects factor and “time” (6 practice blocks) as a within-subject factor to evaluate Day 1’s practice. The effects that 1-Hz rTMS (real or sham) applied over the SMA immediately after practice on Day 1 had on recall on Day 2 with the 2 different practice schedules (random and blocked, groups A–D) were analyzed with a two-way factorial ANOVA design with between-subject factors of practice (blocked vs. random) and rTMS (SMA vs. sham): blocked/sham, blocked/SMA, random/sham, and random/SMA. To gain information on the temporal and spatial specificity of rTMS effects, we included the 5 additional groups described above (E–I). One-way ANOVA was performed for each practice schedule (blocked and random). The values were considered significant if P <0.05.