In 2016 and 2017 we prospectively recruited 30 patients with migraine without aura at the headache clinic of the Sapienza University of Rome Polo Pontino in Latina (Italy). Of these patients, 16 were recorded during the pain-free period (MO), i.e. at a distance of at least 3 days from the last and the next migraine attack at the time of the recording session. The remaining 14 patients were recorded within the first 8 hours of a spontaneous migraine attack, during the headache phase (MI) (see Table 1 for the clinical characteristics). Patients who had received prophylactic therapy in the preceding 3 months or had any other neurological or psychiatric disorder were excluded. For comparison, we recruited 16 healthy volunteers (HV) from the medical and nursing staffs with no personal or family history of migraine or another primary headache. HVs were randomly recorded between patients. All female participants were recorded at mid-cycle at an average of 18.8 (HV), 17.4 (MO) or 17.2 days (MI) after the 1st day of the last menstruation). Clinical information was collected from headache diaries the participants had filled in for at least 1 month before the recording session. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethical Committee of the ‘Sapienza’ University of Rome. All individuals were naïve to the study procedure and provided written informed consent.
Transcranial magnetic stimulation (TMS)
TMS was performed using a MagStim rapid device (MagstimRapid, The Magstim Company Ltd., Whitland, South West Wales, UK) connected to a figure-of-8 coil of which each loop had a 9 cm outer diameter. The coil was placed on the right side of scalp in an optimal position to elicit an electromyographic response in the first dorsal interosseous muscle of the left. The optimal site was labelled with a red dermographic pencil. The motor evoked potential was recorded using silver-chloride cups with the active electrode positioned on the muscle and the reference electrode on the metacarpophalangeal junction of the index finger. The resting motor threshold (RMT) was defined as the minimal intensity needed to evoke an electromyographic response of at least 50 μV with 50% probability in a fully relaxed muscle.
Somatosensory evoked potentials
SSEPs were obtained by electrical stimulation of the left median nerve at the wrist, at an intensity of 1.2 times the motor threshold of the thumb. The active recording electrode was positioned at the C3' position of the 10-20 international system (2 cm posterior to C3), with the reference electrode at Fz; the ground electrode was positioned over the left arm. The electrocortical signals were amplified with Digitimer D360TM pre-amplifiers, recorded by a CEDTM power1401 device (CED Ltd, Cambridge, UK), and off-line analysed with the SignalTM software package version 4.10 (CED Ltd). While subjects were sitting relaxed with open eyes and fixed their attention on the movement of their thumb, 300 non-artefacted traces were acquired at a frequency of 4.4Hz. On the grand-average trace we measured the latency of the parietal components N20 and P25 and their respective peak-to-peak amplitudes. Subsequently, the 300 traces were averaged in 3 blocks of 100 in order to study SSEP habituation. The peak-to-peak amplitude N20-P25 was measured in each block and habituation was calculated as the slope of the regression line along the amplitudes of the 3 sequential blocks.
Somatosensory high-frequency oscillations (HFOs)
To assess thalamocortical activity, we extracted HFOs from the broad-band SSEP signal using the method published elsewhere . In brief, we applied a digital band-pass filter (450-750 Hz, 51 coefficients) on the grand-average SSEP. From the filtered trace we extrapolated the amplitude of the maximum peak of the two oscillation bursts, i.e. the one that occurs before the N20 peak of the broad-band SSEP (early HFOs), and the one that appears after the N20 peak (late HFOs). By identifying source activity from multichannel scalp recordings and the effect of pharmacological agents, previous studies have determined that the early HFO burst reflects thalamic/thalamocortical activity while the late burst is generated by cortical activation [21, 22].
Short-latency afferent inhibition (SAI)
The recordings were performed in the afternoon between 2 P.M. and 7 P.M. while participants were sitting on a comfortable armchair with eyes closed. The peripheral conditioning stimuli on the median nerve (200 ms duration) were applied to the left wrist with a bipolar electrode at an intensity of 1.2 times the motor threshold. The intensity of the TMS test stimulus over the right motor cortex was set at 120% of the resting motor threshold.
We studied SAI using the following protocol. The peripheral electrical stimulus preceded the cortical magnetic stimulus by interstimulus intervals (ISIs) of 2, 4, 6, or 8 msec set with respect to the latency of the SSEP N20 peak in each subject. The baseline MEP was obtained without the conditioning stimulus. For each participant, 40 acquisitions were performed, 5 for each condition (baseline, 2, 4, 6, 8 ms ISIs), randomly applied with a 5-second intertrial interval, and averaged off-line in 5 blocks. For each block, we measured the average peak-to-peak MEP amplitude whereafter we computed the slope of MEP amplitude regression line between the unconditioned and the 4 conditioned recordings as an overall measure of the SAI effect. Three investigators (DDL, CDL, and CA), not involved in patients’ recruitment, performed the recordings. All recordings were anonymized and analyzed blindly off-line by one investigator (F.F).
We used the Statistica for Windows (StatSoft Inc.) version 8.0, for all analyses. An a posteriori sample size calculation based upon a recently published study where SAI was assessed with a similar protocol  showed that 7 subjects per group (standardized effect size of 2.0428) are needed to disclose a significant difference between HV and migraine patients during the attack (power 0.90, alpha error 0.05).
Descriptive analysis showed that MEP peak-to-peak amplitudes of the SAI were not normally distributed. After log transformation, all data reached a normal distribution (Kolmogorov-Smirnov test).
A General Linear Model approach was used to analyze the “between-subjects factor” × “within-subjects factors” interaction effect. The between-subjects factor was the variable “group” (HV, MO, and MI); for SAI the within-subjects factor was “ISI” (baseline, 2ms, 4ms, 6ms, and 8ms) and for SSEP it was “block” (from the 1st to the 3rd block). Two models of repeated measures ANOVA (rm-ANOVA) followed by univariate ANOVAs were employed to investigate the interaction effect. Univariate results were analyzed only if Wilks’ Lambda multivariate significance criterion was achieved.
A regression analysis was used to disclose linear trends in MEP amplitude across ISIs (slope) and in SSEP N20-P25 amplitude across blocks in each group. For SSEP, HFOs, MEP and MEP/SSEP slopes we employed one-way ANOVAs with factor “group” (HV, MO, MI), using least significant difference test for post hoc analysis. P < 0.05 was considered as statistically significant.
Pearson’s correlation test was used to search for correlations between MEP/SSEP amplitude slopes, early/late HFOs and clinical variables such as duration of episodic migraine history, mean monthly attack frequency, mean monthly attack duration, number of days since the last migraine attack, severity of migraine headache on a 0-10 visual analogue scale.