Materials and Methods
Participants
Twenty healthy individuals (15 women, mean age 24.2, ranging 20-29 years, SD 2.8) took part in Experiment 1. Thirteen different healthy individuals took part in Experiment 2. (9 women, mean age 23.8 years, ranging 19-29 years, SD 3.2). They all provided informed consent. They all were screened for any contraindication to TMS (Rossi & Hallett, 2009). The study was approved by the local ethical committee (protocol 2031-032) and was conducted in compliance with the revised Helsinki declaration (Association, 2009).
Localization of TMS Targets
Neuronavigated-TMS was used and the targeted areas were localized by means of individual investigations of anatomical landmarks. In Experiment 1, TMS was delivered to the left mouth-related PMCD as defined in Parmigiani and colleagues (2015). In Experiment 2, TMS was delivered to the face/mouth-related left SMA proper and pre-SMA. In order to localize these areas, we took advantage of Picard and Strick (1996) review (see also Johansen-Berg et al., 2004; Vorobiev, 1998), in which they summarized the results of several positron emission tomography (PET) studies that examined functional activation on the medial wall of humans aimed to providing a common frame of reference for studies of medial wall function (medial portion of BA 6). According to this map, the AC projection crosses exactly in the middle the face area of pre-SMA and SMA proper and therefore to identify this area we used an AC-PC translation of the brain MRIs from each participant and stimulated over the AC projection on the scalp (see Figures xxx xxx).
EMG Recording
Since no primary motor areas were stimulated in these two experiments, no MEPs were collected for further analysis. However, before the experimental session, the right first dorsal interosseous (1DI) was localized and recorded for each subject, and was used to establish the individual resting motor threshold (RMT) in PMCd experiment. In addition, the orbicularis oris (OOr) was recorded in the PMCd experiment in order to exclude an unwanted stimulation of the mouth-related M1. In SMA experiment, the leg-related M1 was functionally localized and recorded in the right tibialis anterior (TA) to have i) the individual RMT and ii) the individual scalp distance from the legM1 hot-spot to the SMA.
TMS
Single-pulse stimulation was achieved by means of a MagPro biphasic magnetic stimulator (Medtronic, Denmark) connected to a MCFB65 coil with 65 mm windings (MagVenture, Skovlunde, Denmark) for REAL stimulation. SHAM stimulation was achieved with a MCF-P-B65 coil with 65 mm windings placebo figure-of-eight coil (MagVenture, Skovlunde, Denmark). Coils were held by an articulated mechanical arm (Manfrotto 244, VitecGroup, Italy). The PMCd coil was positioned with a medio-lateral orientation of the induced current. The SMA coil was positioned tangentially to the midline with the handle positioned caudally. The intensity of the stimulation was around 100% of the RMT of the right 1DI muscle in PMCd experiment and at 100% of the RMT of the right TA muscle in SMA experiment. The stimulation was delivered during the subjects’ RT period of the SST. See the following section for a more detailed description of the stimulations combined to the behavioural task.
Stop-signal paradigm
Before the experiment, participants were tested for their RTs in a simple reaction time task. After a variable foreperiod (600 ms, 1200 ms, 3000 ms, 4500 ms), signalled by a black screen with coloured stars, a pink circle appeared in the centre of the screen (go signal), informing the participants to perform a lifting of the stick, always kept between their lips, from the starting point to the arriving point. Participants were instructed to respond as quickly as possible to the go stimulus. They underwent at least 200 trials of this task. Then, the first, the second and the third quartiles of the single subject RTs were extracted and used in order to define the timing of the TMS pulses and the onset of the STOP signal.
Proper experiment consisted in four blocks of a SST. The SST was composed of a combination of NO-STOP and STOP trials. In the NO-STOP trials, a go signal was presented and participants were instructed to respond as quickly as possible with the mouth movement. In the STOP trials, participants were instructed to stop their response when a stop signal appeared. After a variable foreperiod (2100 ms, 3000 ms, 4500 ms), a pink circle always occurred, informing the participants they were required to move as quickly as possible. In half of the trials, after always the same delay determined by the participant RTs, i.e. the stop signal delay (SSD), a white cross appeared, informing her to inhibit the movement. While participants were required to respond as quickly as possible to the go stimulus in the NO-STOP trials, they were also countermanded to arrest their response on STOP trials, but not wait for the STOP signal to occur (Obeso et al., 2013; Verbruggen & Logan, 2008). On line feedbacks informed participants if they successfully accomplished the NO-STOP or STOP trials (Good!) or failed (Wrong!). In case they took more time than their mean of RTs, they were “pushed” to not get slower with an appropriate feedback (Warning! You are too slow!), on the contrary, in case they did not wait for the go signal to occur, they were warned to do not anticipate (Don’t anticipate!). During the response period, single-pulse TMS was delivered over PMCd (Experiment 1) or SMA (Experiment 2) after the occurring of the go signal. Two possible timing of stimulation were used: at the beginning of the first quartile of the subjects’ RTs (early TMS) or at the beginning of the third quartile (late TMS). The stop signal unvaried across trials, so always occurred at the beginning of the second quartile of single subject’s RTs. Participants underwent four blocks of the task. In two of the blocks participants received a REAL stimulation and in the other two a SHAM stimulation. A block consisted in 60 NO-STOP trials (30 EARLY, 30 LATE) and 60 STOP trials (30 EARLY, 30 LATE) randomly presented, i.e. 120 trials per block, for a total of 240 trials per stimulation (REAL/SHAM). Order of stimulation was balanced across subjects. The trials considered for the analysis was the ones in which TMS, whether REAL or SHAM, were delivered in the pre-movement or early phase of movement. We measured the accuracy of the subjects in both the types of trials, i.e. the ability to conclude the action required when there was a NO-STOP trial as well as being able to inhibit the action when a stop signal was present. We compared REAL vs. SHAM stimulations and we analysed separately data from the two cortical regions (PMCd and SMA). A clarification concerning the balance between NO-STOP and STOP trials in each block as well as the fixed duration of the delay period is necessary. Usually, the performance in the SST is modelled as a race between two processes, the go process on one side, which is triggered by the presentation of the go stimulus, and the stop process on the other side, which is triggered by the presentation of the stop signal (Verbruggen et al., 2009; Logan and Cowan, ?; Aron and Poldrack, 2006). Therefore, when the stop process finishes before the go process, the response is inhibited; otherwise, when the go processes finishes before the stop process, the response is released (see also Duque, 2017). The latency of the stop process is the so called stop-signal reaction time (SSRT) and is generally estimated from a stochastic model. The estimated SSRT is therefore used as the measure of the cognitive control processes that are involved in stopping. In computing the SSRT, also the percentage of the occurrence of the STOP trials could have a role, influencing the response strategy of participants.
In the poresent version of the SST, we decided to introduce a fixed (within subject) duration of the SSD and to present STOP or NO-STOP trials as ruled by chance (50/50).