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All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
- Hot spot and motor threshold:
- Instruct the participant to be at rest- comfortable and with relaxed hands, legs and spinal column.
- Find the hot spot. Target the motor knob at the initial landmark of cortical representation of the abductor pollicis brevis muscle (APB) in motor cortex (M1) and move the coil until there is corresponding APB movement. Use transcranial magnetic stimulation (TMS) intensities evoking motor evoked potentials (MEPs) of around 500 µV over APB. Optimize the coil orientation by changing its angle and tilt to evoke the biggest response over the hot spot.
- Save the coil positioning in the neuronavigator software and reduce the output intensity in steps of 2–3%. Give 10 pulses and if more than 5 out of 10 MEP responses over 50 µV are obtained, then continue reducing the intensity.
- When less than 5 out of 10 responses are evoked, increase the intensity by steps of 1–2%. Motor threshold (MT) is represented as the intensity that produces MEPs larger than 50 µV 5 out of 10 times. The inter-stimulus interval (ISI) for MT should be longer than 1 s, usually set at 3, 4 or 5 s.
- Adjust the intensity using the following steps:
- Start at 120% of MT intensity to produce MEPs over M1 from 500 to 1,500 µV. Record 10 pulses with this stimulator's output so the average response is 1 mV. Increase or decrease the intensity in steps of 1–2% until reaching an average of 1 mV.
- For stimulation intensity, choose the intensity as a percentage of stimulator's output, e.g., 110%, 120%, etc.
- Find the corresponding induced field in V/m (if the system allows). Place the coil over the dorsolateral prefrontal cortex (DLPFC); adjust the stimulator's output until the calculation of the induced field becomes the same as the one over M1 for the same cortical depth.
- Digitize the electroencephalography (EEG) electrodes, so that their position is registered to the brain anatomy.
NOTE: This is a very important step for locating the distribution of neuronal activation and for accurate repositioning of the electrodes in the follow-up session. - Record the TMS-EEG
- Replace the earplugs with the earplugs with air tubes to connect to audio masking if available and add headphones over them. Play the audio masking only during TMS pulse delivery.
- Mount the coil on the coil holder and make sure that the coil does not move or press the electrodes under it. Make sure that the sponge is between the electrodes and the coil.
- Remove all active screens out of the sight of the participant. Give instructions to the participant to stare on a fixed point, not to change his/her head position during TMS delivery and not to blink between the TMS pulses.
- Switch off any fluorescent lights. Run single pulse TMS, short intracortical inhibition (SICI), intracortical facilitation (ICF) and long intracortical inhibition (LICI) in a random order for each participant. Give 100 single and paired pulses. Use various ISI's of 3–4 s (±20%) or a constant of 3–5 s. Give a break of 3–5 min between each condition so the participant can relax and stretch.
NOTE: SICI and ICF involve a paired-pulse TMS paradigm with a subthreshold conditioning stimulus (CS) and a suprathreshold test stimulus (TS). The CS used in this protocol is 80% of MT and the TS at the intensity evoking a 1 mV MEP peak-to-peak. The interpulse interval used for optimal SICI is at 2 ms and for ICF at 12–13. The LICI paradigm involves the pairing of a supra-threshold CS at the intensity evoking the 1 mV MEP peak-to-peak followed by another suprathreshold TS again using the intensity that evoked a 1 mV MEP peak-to-peak and at an inter-pulse interval of 100 ms. The ISI for both single and paired pulse paradigms is determined by the stimulator's charging time, the amount of sessions (longer experiments would require smaller ISI to not overburden the participants) and the analysis that is going to take place. In this study, we used a constant ISI of 5 s due to our stimulator's restrictions and also because we would need several cycles of low frequency band (theta rhythm) for the time-frequency and power spectrum analysis.