Twenty-one healthy right-handed volunteers were initially recruited for the study. However, one subject was excluded due to the low threshold for pyramidal tract activation by cerebellar stimulus, leaving a final sample size of 20 participants (12 females; mean age 28.7±1.3 years).

The sample size calculation was based on a post-hoc analysis using G*Power 3.1 software, assuming an expected effect size (Cohen’s d) of 0.7 in a single group recording at three different time points, resulting in a 0.05 alpha level and a total sample size of 20 to achieve a statistical power of 0.80.

The experimental procedure was approved by the local ethical committee and conducted in accordance with the Helsinki Declaration. All participants gave written consent to participate in the study. None of the participants had a history of neuro-psychiatric diseases or had taken drugs that could interfere with central nervous system activity.

EMG

EMG was recorded from the right first dorsal interosseus (FDI) muscle using 9-mm diameter Ag-AgCl surface electrodes. The active electrode was placed over the muscle belly, the reference electrode at the second finger metacarpophalangeal joint, and the ground electrode over a forearm bony prominence, as previously described [9]. Unrectified EMG signals were recorded (D360, Digitimer Ltd, Welwyn Garden City, UK), amplified (×1000), filtered (bandpass 3–3000 Hz), and sampled (5 kHz) using a 1401 power analog-to-digital converter (Cambridge Electronic Design, Cambridge, UK) and Signal-5 software on a computer and stored for off-line analysis.

SEP

The SEP was recorded following a previous clinical recommendation [10], with the median nerve stimulated using adhesive electrodes placed at the wrist (with the cathode positioned distal). These electrodes were connected to a constant-current stimulator (DS7; Digitimer Ltd.). To ensure that the position of the electrodes was correct, the muscle twitch on the thenar eminence was verified. Stimuli consisted of square wave pulses (0.2 ms duration; 3 Hz frequency; 600 trials; intensity able to evoke a muscle twitch). The N20–P25 waves were recorded by using an active electrode placed at CP3 and a reference electrode placed at Fz on the scalp, following the 10–20 international EEG system. The recorded signal was filtered with a bandpass of 3 Hz to 2 kHz and sampled at a rate of 5 kHz10. The peak-to-peak amplitude of the N20–P25 peak-to-peak wave was measured.

TMS

TMS was delivered using a figure-of-eight coil with an external loop diameter of 7 cm, connected to a Magstim 200 stimulator (Magstim Co., Whitland, Dyfed, UK). The location of the “hot spot” over the FDI M1 representation was carefully identified and marked to maintain the same coil position throughout the experiments. The coil was positioned at 45° angle away from the midline, directed backward and laterally, as reported in previous works [11]. The resting motor threshold (RMT) was determined as the lowest TMS intensity that elicited MEPs of 0.05 mV in at least 5 out of 10 consecutive trials [11].

CBI was measured using a standard paired-pulse TMS paradigm [12,13,14]. Cerebellar stimulation (conditioning stimulus (CS)) was delivered by a double cone coil with an external loop diameter of 7 cm, positioned 3 cm lateral to the inion and contralaterally to M1. To avoid potential artifacts arising from antidromic stimulation of the pyramidal tract, the CS intensity was set at 60% MSO. This precaution was taken by verifying the brainstem activation threshold as described by previous studies [12, 14]. Subjects that showed pyramidal tract activation at 60% of MSO were excluded from the study [15, 16]. Only one recruited subject demonstrated clear pyramidal tract activation and was subsequently excluded from the study. The intensity of the test stimulus (TS) was set at 120% of RMT, and the interstimulus interval (ISI) between the CS and TS was set at 5 ms16.

SAI was obtained by pairing an electrical stimulus applied over the right median nerve (CS), using the same parameters as described for the SEP recording. This was paired with a TMS stimulus (TS) delivered to the contralateral M1, with an ISI of 20 ms [17, 18]. The intensity of the TS intensity was set at 120% RMT. For both CBI and SAI, twelve MEPs were obtained with the TS alone as done in a previous work [19], and twelve MEPs conditioned responses for each ISI were recorded in a randomized order. The test MEP was the same for both SAI and CBI protocols. SAI and CBI were quantified as the ratio between the amplitude of the conditioned MEP and unconditioned MEP.

Experimental Design

The effect of different ISIs of cPAS was evaluated in two experimental sessions performed 2 weeks apart. An additional control experiment was performed only with cPAS25, with at least 1 week separating it from the main experiment.

Experiment 1. Effects of cPAS Intervention at ISIs of 25 ms (cPAS25) and 10 ms (cPAS10) on the Excitability of M1, S1, CBI, and SAI

The cPAS intervention was administered by pairing electrical stimulation of the right median nerve at an intensity three times the subjective perceptual threshold with a cerebellar TMS-pulse set at an intensity of 60% MSO, with an ISI of 25 ms. A total of two hundred pairs of stimuli were administered. MEPs, SEPs, CBI, and SAI were assessed before (baseline), 0 (T0), and 30 min (T30) after cPAS delivery.

In a separate experimental session, the cPAS protocol was delivered as described for cPAS25, but with a 10 ms ISI between ES and cerebellar TMS-pulse, serving as a control condition.

Experiment 2. Effects of cPAS25 on F-wave

To understand the effects of the cPAS25 on the spinal cord, F-waves were recorded in 10 out of 21 participants who participated in experiment 1. Twenty F-waves were collected from the right FDI following subjective supramaximal intensity stimulation of the ulnar nerve at the wrist, which was capable of evoking a compound muscle action potential (CMAP) with the maximum amplitude in the FDI muscle. F-wave persistence was used as a variable and expressed as the ratio between the number of F-wave detectable (amplitude >20 μV) and the total number of recordings [20]. The effects of cPAS25 were assessed by comparing F-wave persistence at baseline, immediately after cPAS25 (T0), and 30 min after cPAS25 (T30).

Statistical Analysis

Data analysis for the MEP, CBI, and SAI protocols utilized the peak-to-peak MEP amplitude. Storing and offline MEP analysis were conducted using Signal 5.0 software on a computer. The ratios of conditioned MEP amplitude to unconditioned MEP amplitude were calculated for both SAI and CBI protocols [10, 15]. Th peak-to-peak N20 SEP amplitude was also measured as a variable measured using Signal 5 software.

Statistical analysis was performed with SPSS 20 software (SPSS Inc., Chicago, IL, USA). All variables were first tested for normality by using the Kolmogorov-Smirnov test. Student’s paired t-test, repeated measures analysis of variance (RM-ANOVA), and planned post hoc t-test with Bonferroni correction for multiple comparisons were used. The compound symmetry of data was evaluated using Mauchly’s test, and when necessary, Greenhouse-Geisser correction was applied. Statistical significance was set at a p-value <0.05. The results are presented as mean ± standard error of the mean (SEM).

Experiment 1

To evaluate whether significant inhibition occurred at baseline in the CBI and SAI protocols and to determine if differences occurred between sessions, a preliminary ANOVA was performed using raw amplitude MEP at baseline as a variable. Consequently, a two-way RM-ANOVA was performed, with cPAS (cPAS10 and cPAS25) and ISI (test MEP; conditioned MEP for CBI and SAI) as within-subjects factors.

Separate two-way RM-ANOVAs were performed on the raw amplitudes MEPs, raw amplitudes of SEPs, and MEP ratios (SAI and CBI) using cPAS (cPAS25, cPAS10) and TIME (baseline, T0, and T30) as within-subjects factors.

Experiment 2

For the evaluation of F-wave persistence, a one-way RM-ANOVA was performed using TIME (baseline, T0, and T30) as the within-subjects factor.