Power Analysis
We conducted an a priori power analysis to determine the required sample size for the experiment. We designed the experiment to have 80% power for detecting the effect sizes that we previously found for the effect of motivational goal-priming on the motor system (0.64, Cohen’s d) and motor action (0.50–0.82) (13) and/or pupil diameter (0.61, Cohen’s d) (16), using a significance level of 5%. We used G*Power 3.1® (Institut für Experimentelle Psychologie, Düsseldorf, Germany) to compute the required total sample size of the current study by conducting a repeated-measures analysis of variance (ANOVA) with within factors (Control or Priming or Priming+Reward), using 95% power (1-β error probability). The computed required sample size was 12 participants.
Participants and Procedures
Seventeen healthy Japanese right-handed individuals, evaluated using the Edinburgh Handedness Inventory (17), participated in the study. The participants were 15 men and two women, with a mean age ± standard deviation of 19.3 ± 0.95 years. All participants provided both written and verbal informed consent. The study was conducted in accordance with the Declaration of Helsinki. We confirmed that pregnant women were not among our participants to avoid the unknown effects of TMS on an unborn fetus. All participants were university students who reported no strength training history, which indicated that they had not received training in production of the maximal force generated briefly by a muscle or group of muscles. The experimental procedures complied with relevant laws and institutional guidelines and were approved by the Human Research Ethics Committee of the Faculty of Sport Sciences of Waseda University (Approval Number: 2020-411).
The experiments were designed to examine the effects of motivational goal-priming on sustained handgrip maximal voluntary force, pupillary size, and MEPs in the flexor carpi ulnaris (FCU) muscle in response to TMS (see TMS for details). Each experiment consisted of two tasks (pre-sustained MVC and sustained MVC), which were spaced at least 3 min apart on the same day. The total experimental period exceeded 90 min. In the pre-sustained MVC task as a baseline of the sustained MVC task, participants performed three brief MVCs (1–2 s duration) when an experimenter gave a cue (“One, two, three, squeeze”) with a 60 s inter-squeeze interval. After a rest of approximately 3 min, participants performed a sustained MVC according to experimental instructions displayed on a screen in front of them (see Pupil Diameter Measurement for details). Visual feedback of handgrip force was not given throughout the sustained MVC. The sustained MVC task was performed under three conditions, each lasting approximately 237 s (Fig. 1A). Participants were not given advance notice of when the sustained MVC condition would terminate. The following three conditions were tested: priming words related to physical exertion paired with subsequently displayed “reward” words (priming-plus-reward condition); priming words paired with non-reward words (priming condition); and no priming words (control condition). Participants experienced all three conditions in a random order with a more than 30 min interval between conditions, which was expected to enable central fatigue to recover (18, 19). The numbers of participants in the six possible combinations of the execution order were as follows: (control, priming, priming-plus-reward) 2; (control, priming-plus-reward, priming) 2; (priming, control, priming-plus-reward) 3; (priming, priming-plus-reward, control) 3; (priming-plus-reward, control, priming) 3; (priming-plus-reward, priming, control) 4. To investigate motor cortex excitability, TMS was applied to the left M1 1.5 s after the positive or neutral word disappeared (see Priming procedure for details). Thus, 50 MEPs were obtained for each condition. Under all conditions, participants were asked to keep their heads still and to keep their hands on their lap in a sitting posture while maintaining as much stability in the core as possible, and to keep viewing the screen in front of them.
Testing of Subliminal Stimuli
To confirm that the subliminal primed words were not consciously perceived,
we conducted a separate experiment in which different participants (36 males and eight females; mean age ± SD: 22.0 ± 2.0 years) completed the subliminal and supraliminal conditions. Participants were asked to indicate whether they saw a Japanese verb related to physical exertion. The post-masked subliminal exertion-related primes were attended, but not reported. Response accuracy was 50.09% ± 0.94%, indicating that their judgments did not differ from chance, and that they could not see the priming words.
Priming Procedure
To manipulate unconscious goal pursuit, we adopted an experimental procedure used
in previous studies (13, 16) (Fig. 1B). Specifically, we used five Japanese verbs related to physical exertion (“to exert” [hakki-suru], “to struggle” [funtou-suru], “to work hard” [mogaku], “to energize” [sei wo dasu], and “to strive” [doryokusuru]) as motor goals, five positive adjectives (“nice” [suteki na], “great” [subarashii], “fantastic” [kibunsaikouno], “satisfactory” [manzoku na], and “enjoyable” [tanoshii]) as positive reward, and five neutral adverbs (“almost” [hotondo], “at least” [sukunaku tomo], “finally” [saigoteki ni], “nearly” [hobo], and already [sude ni]) as non-reward words. In the priming-plus-reward condition, for 25 of the 50 trials, barely visible presentation of one of the five exertion words was followed by fully visible presentation of one of the five positive words. For the remaining 25 trials, barely visible presentation of a random letter string was followed by fully visible presentation of one of the five neutral words. Thus, in this condition, the barely visible exertion primes were always paired with positive words. In the priming condition, the exertion primes were paired with neutral words (25 trials), and the random letter strings were paired with positive words (25 trials). Thus, in this condition, although exertion primes and positive words were both displayed, they were never paired with each other. In the control condition, only random letter strings were used as primes and were paired with positive words on 25 trials and with neutral words on 25 trials; barely visible exertion words were never displayed. In this way, the viewing of positive and neutral words was balanced at 25 trials each for all conditions. The order of possible stimulus pairs was randomized within each condition.
Each trial in each condition began with a 1000 ms presentation of five different strings of eight pseudorandom letters (DZXLTOTM, YSTZBXTU, VCFTHYPC, CBEXGTVY, and ZTAWYDBH) as a forward mask (Fig. 1B). This was followed by the barely visible prime, displayed for 33 ms. One randomly selected random letter string among those five was again displayed for 100 ms as a backward mask, after which a consciously visible word was presented for 150 ms. Occasionally, a dot was presented for 33 ms (visible because of the absence of a backward mask), either above or below the neutral or positive word. Participants were instructed to see a dot, to bring the post-masked barely visible primes to their attention. Trials occurred every 3.5 s within each condition. We used a 60 Hz CRT screen to display the words, and the experimental procedure was created with software designed for psychological experiments (Inquisit 3 Desktop Edition, Millisecond Software, Seattle, WA, USA).
Pupil Diameter Measurement and Analysis
To measure the pupil diameter, we adopted a method to examine the effects of unconscious goal pursuit on the pupil-linked neuromodulatory system state in our previous studies (15, 16). The pupil diameter was measured using a TalkEye Lite system (Takei Scientific Instruments Co., Ltd., Tokyo, Japan). An image around the pupil was obtained using a camera employing near-infrared light-emitting diodes and a video graphics array (640 × 480) (built-in digital signal processor) camera module (NCM03-V, Nippon Chemi-Con Corporation, Tokyo, Japan). Banalization processing was performed on each image, and the pupil diameter was then measured according to the methods described by Wang et al. (20). Changes in pupil size were estimated by the area of the pupil (15, 16) while participants viewed the screen in front of them under all conditions. We calculated the average pupil area (dots) for 500 ms before each TMS (see TMS) during squeezing of a handgrip device (see Handgrip Force Measurement) under the three conditions of the sustained MVC task.
The following steps were taken to exclude the effect of experimenter expectations on participant responses and measurements as much as possible, and to objectively estimate the effect of motivational goal-priming under the sustained MVC task. 1) The experimental procedure of the sustained MVC task was conducted automatically using a 60 Hz cathode ray tube screen to display the text, and the experimental procedure was created using software designed for psychological experiments (Inquisit 3 Desktop Edition, Millisecond Software, Seattle, WA, USA). 2) All participants were instructed to follow starting and stopping signals on the screen. 3) Pupil diameter measurements were automatically performed using a specially designed device with an eye-capturing camera to obtain an image around the pupil. Consequently, the paradigm used in the present study was less susceptible to experimenter bias compared with outcome measurements that have typically been used for examining maximal voluntary force in previous studies (21).
All word stimuli were displayed in black (22.5 cd/m2: the mean value of five measurements of luminance with an LS160 luminance meter; Konica Minolta, Inc., Tokyo, Japan) on a white screen (124.3 cd/m2) in the experimental procedure. Immediately before the word presentation, the color of the screen was momentarily white without any black words. The pupil diameter may have transiently decreased because of the increase in luminance caused by the white screen with a maximum luminance of 129.6 cd/m2. Thus, we were unable to completely eliminate the possibility that this transient change in luminance affected the pupil diameter. However, any effect on the results would likely be minimal because this phenomenon was present for all participants and conditions.
Handgrip Force Measurement and Analysis
The voluntary force was measured using a handgrip device (KFG-5-120-C1-16, Kyowa Electronic Instruments, Tokyo, Japan). Under the pre-sustained MVC conditions, participants performed five brief MVCs (1–2 s duration) on a cue given by an experimenter (“One, two, three, squeeze”) with a 30 s inter-squeeze interval. Under the sustained MVC condition, the experimenter asked participants to squeeze the handgrip device with the right (dominant) hand with maximum effort when some kind of the priming word appeared on the display, and to stop squeezing when the word disappeared. The handgrip device was fixed to the right thigh with an elastic band so that the device did not move when it was squeezed by the participant. The maximal values of the exerted force were averaged from the 500 ms steady state of the force curve before each TMS (Fig. 2A) across the three trials for the pre-MVC condition with three brief MVCs, and across the 50 trials for the sustained MVC condition with 50 MEPs (see TMS for details). These averages were taken as the handgrip maximal voluntary force. To estimate the relative levels of responsiveness of M1 to voluntary drive during sustained maximal voluntary contractions, force produced by the superimposed twitch (twitch force) following TMS was expressed as a fraction of the pre-stimulus force which was the 500 ms steady state of the force curve before each TMS (Fig. 3), on the basis of a previous study (4).
TMS
To measure MEP, we adopted a method to examine the effects of unconscious goal pursuit on the motor system state in the previous study (13). In the pre-sustained MVC and the sustained MVC tasks, single-pulse TMS was administered via a stimulator (M2002, Magstim, Whitland, UK) using a double-figure-eight-shaped coil (4150-00 Double 70 mm Alpha Coil, Magstim) with a maximum magnetic field strength of 1.55 T. Each participant sat upright with their elbows bent in front of them, and their hands resting on their thighs. The TMS coil was then positioned over the finger area of the left M1, which was determined as the area with the lowest resting motor threshold (rMT). This was defined as the area for which MEPs with peak-to-peak amplitudes greater than 50 µV were induced in the FCU muscle (22, 23) in at least five of 10 trials when participants were fully relaxed with their eyes closed (24). During MEP recording, participants were asked to remain in a resting state. The coil position was stabilized throughout the experiment using a coil stand made from multiple products (Manfrotto Distribution KK, Tokyo, Japan). The optimal scalp position of M1 was marked directly onto the scalp with a black marker pen. The positioned coil was monitored continuously to maintain consistent positioning throughout the experiment. The rMTs ranged from 40% to 70% of the maximum stimulator output. Stimulation was manually delivered three times over the target site during each brief MVC (1–2 s duration), with a 60 s inter-squeeze interval in the pre-sustained MVC task (Fig. 2A, B). The stimulation was automatically delivered 50 times in the sustained MVC task, the stimulus intensity of which was set from 60% to 80% of the maximum stimulator output for each participant during handgrip force exertion (Fig. 3). Thus, MEPs in the sustained MVC task were recorded 50 times. Surface electromyography (EMG) was obtained from the right FCU muscles via bipolar silver surface electrodes (10 mm in diameter) using the tendon-belly method (23). The skin overlying the identified muscles was cleaned with alcohol pads prior to electrode placement. Signals (analysis time of 30 ms) were amplified using a bandpass filter (15 Hz–10 kHz) and digitized (MEG-6108; Nihon Kohden Co., Tokyo, Japan) at a sampling rate of 4 kHz.
Background EMG and MEP Measurement and Analysis
We measured the peak-to-peak amplitude of each MEP, the size of which reflects corticospinal excitability (25, 26) (Fig. 2C). We calculated the averaged waveform of the MEP under the pre-sustained MVC (an average of 3 recordings) and sustained MVC tasks (an average of 50 recordings) (see TMS for details). To measure the EMG background (bEMG), a rectified EMG signal having a period of approximately 100 ms before TMS was integrated, with the force kept at the maximum force level (Fig. 2A, B). These analyses were performed using analysis software (LabChart 7.3.8; ADInstruments, Tokyo, Japan).
The duration of the silent period was taken as the time interval from the stimulus artifact to the return of continuous EMG (27, 28) (Fig. 2C). When it was difficult to determine the end of the silent period (because voluntary EMG activity recovers gradually rather than abruptly), the end of the silent period was determined as the moment that the corresponding rectified EMG activity reached a value within two standard deviations of the rectified EMG signal in the period approximately 100 ms before TMS (29, 30).
Statistical Analysis
The maximal voluntary force, twitch force, the duration of the silent period, MEP, bEMG, and pupil area under the sustained MVC task were analyzed in a repeated-measures one-way ANOVA of the experimental group (within-participants factor: [Control or Priming or Priming+Reward]). Greenhouse–Geisser corrections were applied when appropriate to adjust for non-sphericity, changing the degrees of freedom using a correction coefficient. A significance threshold of p < 0.05 was chosen for all tests. When the results of the main effect and interaction of the one-way ANOVA are presented, partial η2 (ηp2) was also shown as an effect size index. ηp2 values of 0.1–0.24, 0.25–0.39, and ≥ 0.4 were considered to reflect small, medium, and large effects, respectively (31). Pearson’s correlation analysis was performed to determine the associations between the duration of the silent period and twitch force, and between the maximal voluntary force and pupil area.