Abstract

We designed a new paired associative stimulation (PAS) protocol that combines experimental pain evoked by laser stimuli and transcranial magnetic stimulation (TMS) (Laser-PAS) to primary motor cortex (M1). We tested in healthy subjects whether Laser-PAS elicits cortical plasticity as reflected by long-term changes in motor-evoked potentials (MEPs) (after-effects). In separate experiments, we examined numerous variables including changes induced by varying the interstimulus intervals (ISIs) and Laser-PAS-induced changes in target and non-target muscle MEPs. We measured MEPs after repetitive laser or TMS (rTMS) pulses, and compared magnetic- and electric (TES)-induced MEPs. We tested MEPs after applying Laser-PAS with laser pulses ipsilaterally to M1. Finally, we studied subjects receiving an N-methyl-d-aspartate (NMDA) receptor antagonist (memantine) or placebo (α-lipoic acid). During Laser-PAS at the 50 ms ISI MEPs decreased, thereafter they increased for 60 min; other ISIs induced no after-effects. The after-effects remained restricted to the target muscle. Repetitive laser pulses and rTMS induced no after-effects. After Laser-PAS, TMS-induced MEPs increased, whereas TES-induced MEPs did not. Laser-PAS with laser pulses ipsilaterally to M1 left MEPs unchanged. Memantine, but not α-lipoic acid, abolished the after-effects. In conclusion, Laser-PAS elicits NMDA-dependent cortical plasticity and provides new insights into human pain-motor integration.

Introduction

Heat-evoked experimental pain is increasingly evoked in humans with the laser-evoked potential (LEP) technique. LEPs comprise a small lateralized negative component (N1) and a later negative–positive complex (N2–P2) (Bromm 1985; Treede 1995). Although the primary somatosensory area contributes (Iannetti and Mouraux 2010; Valentini et al. 2012), the LEP N1 component predominantly arises from the secondary somatosensory area (SII) and posterior insula (operculoinsular region), whereas the N2–P2 component reflects anterior cingulate cortex (ACC) and bilateral insular activation (Spiegel et al. 1996; Bromm and Lorenz 1998; Garcia-Larrea et al. 2003; Cruccu et al. 2008).

Experimental pain in humans affects the primary motor cortex (M1) through physiological processes increasingly recognized as pain-motor integration (Svensson et al. 1995, 2004; Valeriani et al. 1999, 2001; Raij et al. 2004; Adachi et al. 2008). To date, only one study has investigated how heat-evoked experimental pain influences M1 excitability as tested by transcranial magnetic stimulation (TMS) (Valeriani et al. 1999). When a single TMS pulse followed laser stimulation at the LEP N1 + 50 ms interstimulus interval (ISI), motor-evoked potentials (MEPs) decreased reflecting M1 inhibition (Valeriani et al. 1999).

Besides changes in overall M1 excitability, repetitive TMS (rTMS) techniques can now investigate rTMS-induced MEP changes (after-effects) reflecting long-term potentiation (LTP)- and depression (LTD)-like plasticity in human M1. The original paired associative stimulation (PAS) protocol combines repetitive cortical and peripheral nerve electric stimulation. When cortical stimulation follows nerve stimulation at the wrist at the 25-ms ISI, MEPs increase owing to LTP-like, whereas at the 10-ms ISI, they decrease owing to LTD-like plasticity. Some evidence attributes PAS-induced plasticity to sensorimotor integration processes (Stefan et al. 2000, Ziemann et al. 2008).

Given the crucial role of M1 LTP/LTD in motor control (Asanuma and Keller 1991; Iriki et al. 1991; Classen et al. 1998; Rioult-Pedotti et al. 1998, 2000; Muellbacher et al. 2000; Ziemann et al. 2008), knowing whether heat-evoked experimental pain elicits M1 LTP/LTD-like plasticity should help us to explain the mechanisms underlying human pain-motor integration.

In this study, seeking information on human pain-motor integration, we designed a new PAS protocol, laser-paired associative stimulation (Laser-PAS), combining laser skin pulses that selectively activate the nociceptive system, and TMS. In healthy subjects, we investigated M1 plasticity as reflected by long-term changes in MEPs during and for 90 min after Laser-PAS (after-effects) at LEP N1 + 50 ms ISI. We examined several variables including effects induced by varying ISIs and investigated whether Laser-PAS-induced MEP changes involved target and non-target muscles (topographic specificity). To clarify whether MEP amplitude changes depend on repetitive laser pulses or rTMS, we compared the after-effects induced by Laser-PAS and repetitive laser pulses or rTMS given alone. To verify whether mechanisms underlying Laser-PAS reflect changes in cortical or subcortical excitability, we collected TMS-induced MEPs (reflecting changes in cortical excitability) and MEPs to transcranial electric stimulation (TES) (reflecting changes in cortico-spinal axon excitability). We also tested MEP amplitude changes after applying Laser-PAS ipsilaterally to the stimulated M1. Because M1 LTP/LTD involves N-methyl-d-aspartate (NMDA)-dependent glutamatergic transmission, to investigate whether Laser-PAS implies NMDA receptor activation, we designed a randomized, double-blind, placebo-controlled, cross-over study in subjects receiving an NMDA antagonist (memantine) or placebo (α-lipoic acid).

Materials and Methods

Subjects

The study group comprised 17 right-handed healthy subjects (7 men and 10 women; mean age ± SD: 27 ± 3 years, age range 24–35). Subjects gave their informed consent. The study was approved by the institutional review board and conformed with the Declaration of Helsinki.

Laser Stimulation Technique and LEP Recordings

Laser stimuli were delivered with a neodymium:yttrium–aluminium–perovskite laser stimulator (Nd:YAP, wave length 1.34 μm, pulse duration 2–20 ms, maximum energy 7 J) (EL.EN, Florence, Italy) under fiber-optic guidance. We activated Aδ-fibers by delivering laser stimuli over the right-hand dorsum in the ulnar region. To avoid habituation, damage to the skin, fatigue and nociceptor sensitization, the laser beam was slightly shifted after each stimulus and ISIs were varied pseudorandomly (10–15 s). To determine the laser perceptive thresholds (heat and pain thresholds), we delivered series of 3 stimuli at increasing and decreasing intensities, and defined thresholds as the lowest intensities at which the subjects perceived heat and painful sensations in at least 50% of the trials. Each subject rated pain perception on a 0–10 point numerical rating scale (pain rating) (Perchet et al. 2008). To evoke clear and stable LEPs, we delivered laser pulses at the intensity able to elicit painful pinprick sensations related to Aδ-fiber activation (corresponding to a level of at least 4 in the pain rating scale) (Perchet et al. 2008). Laser intensity was kept stable throughout the experiment. After LEP recordings each subject again rated pain perception. LEPs were recorded according to the recommendations issued by the International Federation of Clinical Neurophysiology (Cruccu et al. 2008). In brief, LEP responses were recorded through paired surface electrodes (Ag–AgCl) placed at the T3, T4, Cz, and Fz according to the International 10–20 System and referenced to the nose. An additional electrode, positioned over the Fpz, was used for grounding. To check possible contamination by eye movement and blink, we also recorded the electroculogram. Electrode impedance was kept below 5 kΏ throughout the experiment. Signals were recorded, amplified, and filtered (bandwidth 0.5–50 Hz) with a Digitimer D360 (Digitimer Ltd, Welwyn Garden City, United Kingdom), acquired at a sampling rate of 5 kHz through a 1401 plus A/D laboratory interface (Cambridge Electronic Design, Cambridge, United Kingdom) and stored on a personal computer for off-line analysis (Signal software; Cambridge Electronic Design, Cambridge, United Kingdom). We averaged 15 artifact-free trials (uncontaminated by blink-related electromyographic (EMG) activity). Two LEP components were measured: the left N1 component and the N2–P2 complex latency; N1 amplitude was measured from the isoelectric line to peak, and N2–P2 amplitude peak-to-peak. LEPs were recorded 30 min before TMS and MEP recordings started.

TMS and MEP Recordings

TMS was delivered through a repetitive magnetic stimulator (Magstim Super Rapid—The Magstim Company Ltd, Whitland, United Kingdom) connected to a figure-of-eight coil (external wing 9 cm in diameter) placed tangentially to the scalp on the left hemisphere, with the handle pointing back and away from the midline at 45° inducing postero-anterior and antero-posterior (PA–AP) biphasic currents in the brain. The coil was placed over the optimum scalp position (hot spot) to elicit MEPs in the abductor digiti minimi (ADM) muscle of the right hand. To ensure that the stimulating coil remained in a constant position throughout the experiments, the hot spot was marked on the scalp with a soft-tipped pen. Motor threshold was determined at rest (RMT) as the lowest intensity able to evoke an MEP of more than 50 μV in at least 5 of 10 consecutive trials in the ADM muscle (Rossini et al. 1994). RMT was determined in steps of 1% maximum stimulator output intensity. EMG activity was recorded through a pair of surface electrodes (Ag/AgCl) placed over the right ADM muscle, using a belly-tendon montage. EMG was recorded, amplified, and filtered (bandwidth 5–1 kHz) with the same Digitimer D360 and stored with the same apparatus already described for LEP recording. The baseline EMG activity level before, during and after TMS was controlled by visual-feedback through an oscilloscope screen and auditory feedback through a loudspeaker. To exclude possible confounding effects from involuntary muscular contraction, trials with background EMG activity were rejected. MEP amplitudes were measured peak-to-peak (mV) and then averaged.

Experimental design

The study comprised Experiments 1–8 (Fig. 1). All 17 healthy subjects participated in Experiment 1, whereas different subgroups pseudorandomly participated in Experiments 2–8. All experimental sessions took place at comparable daytime and at least 1 week elapsed between each session. Subjects were tested fully relaxed and with their eyes open.

Figure 1.

Experimental paradigm. In the first set of experiments, we tested whether a 10-min series of Laser-PAS50 (L-PAS50) induced changes in motor-evoked potential (MEP) amplitude. We measured the time course of MEP modulation during Laser-PAS50 and for 10–90 min after Laser-PAS50 ended (Experiment 1). We investigated the effect of varying the interstimulus intervals from 0 to 200 ms (Experiment 2). We tested changes in MEP amplitude recorded from the target abductor digiti minimi (ADM) as well as from non-target abductor pollicis brevis (APB) muscles (Experiment 3). We investigated possible changes in MEP amplitudes after 0.1 Hz repetitive laser stimuli (Experiment 4), and repetitive transcranial magnetic stimulation (rTMS) given alone (Experiment 5). After Laser-PAS50, we also tested possible changes in MEPs evoked by transcranial electric stimulation (TES) (Experiment 6). We compared MEPs before and after Laser-PAS50 implying laser stimuli given contralaterally and ipsilaterally (ipsilateral L-PAS50) to the stimulated primary motor cortex (M1) (Experiment 7). Finally, Experiment 8 consisted in a randomized, double-blind, placebo-controlled, cross-over study designed to investigate the effect Laser-PAS50 on MEP amplitudes in subjects receiving memantine or α-Lipoic acid. All experimental sessions began with laser-evoked potential (LEP) recording.

Figure 1.

Experimental paradigm. In the first set of experiments, we tested whether a 10-min series of Laser-PAS50 (L-PAS50) induced changes in motor-evoked potential (MEP) amplitude. We measured the time course of MEP modulation during Laser-PAS50 and for 10–90 min after Laser-PAS50 ended (Experiment 1). We investigated the effect of varying the interstimulus intervals from 0 to 200 ms (Experiment 2). We tested changes in MEP amplitude recorded from the target abductor digiti minimi (ADM) as well as from non-target abductor pollicis brevis (APB) muscles (Experiment 3). We investigated possible changes in MEP amplitudes after 0.1 Hz repetitive laser stimuli (Experiment 4), and repetitive transcranial magnetic stimulation (rTMS) given alone (Experiment 5). After Laser-PAS50, we also tested possible changes in MEPs evoked by transcranial electric stimulation (TES) (Experiment 6). We compared MEPs before and after Laser-PAS50 implying laser stimuli given contralaterally and ipsilaterally (ipsilateral L-PAS50) to the stimulated primary motor cortex (M1) (Experiment 7). Finally, Experiment 8 consisted in a randomized, double-blind, placebo-controlled, cross-over study designed to investigate the effect Laser-PAS50 on MEP amplitudes in subjects receiving memantine or α-Lipoic acid. All experimental sessions began with laser-evoked potential (LEP) recording.

Experiment 1: Effect of Laser-PAS on MEP amplitude

In this experiment, designed to investigate whether the new Laser-PAS protocol directly induced changes in MEP amplitude, we first delivered 20 single TMS pulses able to elicit MEPs of 1 mV from the right ADM at baseline (T0) (corresponding to about 120% RMT). We recorded and measured a clear-cut peak LEP N1 component according to the international recommendations (Treede et al. 1999; Cruccu et al. 2008; Perchet et al. 2008). We then applied conditioning Laser-PAS consisting of 60 rTMS pulses at 0.1 Hz, each TMS pulse following a single laser stimulus delivered at an ISI of LEP N1 latency + 50 ms (total duration of intervention: 10 min) (Laser-PAS50). The specific 50 ms laser-TMS interval was set as described by others (Valeriani et al. 1999). Laser pulses were delivered at an intensity twice the laser perceptive threshold. To investigate the time course of possible after-effects induced by Laser-PAS, we compared baseline MEPs (T0) with those recorded immediately after Laser-PAS ended (T1) and at 10 (T2), 20 (T3), 30 (T4), 40 (T5), 50 (T6), 60 (T7), 70 (T8), 80 (T9), and 90 min (T10) after it ended. To verify possible changes in MEP amplitudes during Laser-PAS, we also compared baseline MEPs at T0 with the 60 MEPs collected during Laser-PAS and averaged them at 3 time points: from 1 to 20 (T0a), from 21 to 40 (T0b), and from 41 to 60 (T0c). All 17 subjects were studied at T1–T7 and in 5 subjects we prolonged MEP collection from T8 to T10.

Experiment 2: Effect of Varying ISIs

To test how different ISIs influenced MEP amplitude changes induced by Laser-PAS in MEP amplitude, we randomly delivered Laser-PAS at N1 + 0, 50, 100, and 200 ms in the same group of subjects in 4 separate sessions with at least 1 week elapsing between each session. As we did for Experiment 1, we recorded 20 MEPs before (T0) and after conditioning (T1–T7). Five subjects participated in this experiment.

Experiment 3: Topographic Specificity

Experiment 3 was designed to see whether changes in MEP amplitude after Laser-PAS50 remain topographically restricted to the target ADM muscle, or spread to non-target muscles including abductor pollicis brevis (APB). In this experiment, we first calculated the intensity able to elicit 1 mV MEPs from the right ADM and APB muscles at T0, and then collected 20 MEPs from both muscles before (T0) and after conditioning (T1–T7). Laser-PAS50 was given with the same procedure as described in Experiment 1. Five subjects participated in this experiment.

Experiment 4: Effect of Repetitive Laser Pulses on MEP Amplitude

To determine whether possible Laser-PAS50-induced changes in MEP amplitudes simply reflect laser-induced after-effects, we evaluated MEPs after delivering 60 laser pulses at 0.1 Hz and at the same intensity used for Laser-PAS50. We recorded 20 MEPs before (T0) and after conditioning (T1–T7). A subgroup of 5 subjects participated in this experiment.

Experiment 5: Effect of rTMS on MEP Amplitude

To verify whether possible changes in MEP amplitudes after Laser-PAS50 depend on Laser-PAS50, or simply reflect rTMS-induced after-effects, we evaluated MEPs after delivering rTMS alone, at the same frequency and with the same number and intensity of pulses used in Laser-PAS50. To do so, we applied conditioning stimulation consisting in a single 0.1 Hz-rTMS train of 60 pulses delivered at the same intensity used for evoking 1 mV MEPs at T0. We recorded 20 MEPs before (T0) and after conditioning (T1–T7). A subgroup of 5 subjects participated in this experiment.

Experiment 6: Cortical versus Subcortical Mechanisms

In Experiment 6 to investigate whether possible Laser-PAS50-induced changes in MEP amplitudes depend on cortical or subcortical interactions between laser pulses and TMS, we used the TES technique. Unlike PA–AP biphasic TMS, anodal TES is thought to elicit MEPs predominantly by directly activating cortico-spinal axons (Day et al. 1987; Petersen et al. 2003; Di Lazzaro et al. 2004). According to a previously reported technique (Petersen et al. 2003; Di Lazzaro et al. 2004), we delivered anodal TES through a pair of surface electrodes (Ag/AgCl) placed over the scalp with the anode over M1 and the cathode over the vertex. TES was delivered with a Digitimer DS7 constant-current stimulator by applying square-wave stimuli with a pulse width of 1 ms. We first determined the electric RMT (RMTelectric) and then calculated the intensity able to elicit MEPs of 1 mV in amplitude (corresponding to about 120% RMTelectric). Twenty MEPs from ADM muscle were randomly elicited by single TMS or anodal TES pulses before at T0 and at T1–T7 after conditioning. A subgroup of 5 subjects participated in this experiment.

Experiment 7: Effect of Laser-PAS with Laser Pulses Given Ipsilaterally to M1

Experiment 7 was designed to determine whether Laser-PAS50 with laser pulses given ipsilaterally to the stimulated M1 elicits after-effects. Accordingly, we delivered Laser-PAS50 as described in Experiment 1 over the left-hand dorsum in the ulnar region. We recorded 20 MEPs before (T0) and after conditioning (T1–T7). A subgroup of 5 subjects participated in this experiment.

Experiment 8: Effect of a Drug Acting on the NMDA Receptor

To investigate whether a drug acting on the NMDA receptor modulates Laser-PAS50–induced changes in MEP amplitudes, we designed a randomized, double-blind, placebo-controlled, cross-over study in subjects receiving the NMDA antagonist (memantine) or placebo (α-lipoic acid) just before Laser-PAS50. A subgroup of 8 subjects participated in 2 separate sessions. One of the authors not directly involved in the experiments or data analysis planned each experimental session and delivered the drug. All the other investigators and subjects involved in this experiment remained blinded. According to the experimental design previously used by Huang et al. (2007), subjects received the first 5 mg of memantine (or placebo) before night-time sleep 2 days before the experiment and, on the second day, took 5 mg at 8:00 AM and 8:00 PM. Finally, on the third day, subjects received memantine (10 mg) (or placebo) at 8 am and Laser-PAS50 was delivered at 10 am. Placebo consisted in a challenge with 10 mg α-lipoic acid, a drug predominantly acting as free radical scavenger and antioxidant (Packer et al. 1995; Smith et al. 2004). We recorded 20 MEPs before (T0) and after conditioning (T1–T7).

Statistical Analysis

Data collected in all experimental sessions were analyzed as absolute values (mV) by repeated measures analysis of variance (ANOVA). To test possible changes in MEP amplitudes and RMT after Laser-PAS, we used 1-way ANOVA with “Time” as the main factor of analysis in Experiments 1. The factor “Time” consisted in T0 versus T1, T2, T3, T4, T5, T6, and T7 in the whole group of 17 subjects, and also T8, T9, and T10 in the subgroup of 5 subjects. To verify possible changes in MEP amplitudes during Laser-PAS, we also used 1-way ANOVA with factor “Time” consisting in T0 versus T0a, T0b and T0c. In the remaining experiments (2, 3, 4, 5, 6, 7, and 8) we used a 2-way ANOVA with the factors “ISI” and “Time” (Experiment 2), “Muscle” and “Time” (Experiment 3), “Stimulation” and “Time” (Experiment 4, 5 and 7), “Cortical vs subcortical activation” and “Time” (Experiment 6), and “Drug” and “Time” (Experiment 8). To compare RMTelectric at all the time points considered (T0 vs. T1–T7) in Experiment 6, we also used 1-way ANOVA with factor “Time”. One-way ANOVA with factor “Conditions” was used to compare the RMT, and the intensity required to evoke MEPs at T0 in all experimental sessions. Finally, non-parametric 1-way ANOVA (Kruskal–Wallis Test) with factor “Conditions” was used to compare the laser perceptive threshold and pain rating collected in each subject in all experiments.

The Spearman correlation test was used to assess possible correlations in each subject between possible Laser-PAS50-induced changes in MEP amplitudes and laser perceptive threshold and pain rating, RMT, RMTelectric, and the intensity required for evoking 1 mV amplitude MEPs at T0.

Tukey honest significant difference test was used for all post hoc analyses. The Greenhouse–Geisser correction was used when necessary to correct for non-sphericity. A P-value <0.05 was considered significant for all statistical analyses.

Results

None of the subjects experienced any adverse effects during or after laser stimulation, TMS, memantine or placebo (including sedation or inattention). All subjects perceived laser stimuli as painful in all experimental sessions (Table 1).

Table 1.

Analytic results for laser-evoked potential (LEP) recordings and pain rating in the 17 healthy subjects

Subjects Heat threshold Pain threshold Stim. int. (mJ/mm2Pain rating N1 lat. (ms) N1 amp. (μV) N2 lat. (ms) N2 amp. (μV) P2 lat. (ms) P2 amp. (μV) 
76 102 203 120 4.4 206 17.0 377 12.0 
51 178 203 122 4.4 172 7.9 218 4.7 
76 178 203 153 5.0 206 4.5 331 5.6 
51 76 203 126 7.3 167 5.4 239 4.4 
102 127 203 189 2.3 210 4.5 296 3.4 
76 102 178 106 7.4 184 9.6 241 5.8 
51 102 102 190 13.8 214 22.5 309 24.3 
76 178 203 185 1.6 240 3.3 380 7.2 
51 76 178 157 3.9 203 3.0 260 3.9 
10 76 102 254 168 3.9 200 4.3 331 7.2 
11 51 76 178 200 2.5 250 6.5 360 5.0 
12 102 127 178 161 4.4 198 5.3 267 14.8 
13 21 76 178 136 1.3 186 9.0 253 8.0 
14 76 102 127 170 5.4 190 14.0 281 21.0 
15 51 76 102 175 3.3 208 27.6 281 17.0 
16 76 102 127 161 4.2 185 4.6 240 6.8 
17 51 127 178 170 11.0 179 6.9 272 9.8 
AV 66 112 176 158 5.1 200 9.2 290 9.5 
SD 20.7 36.1 40.5 1.3 27.6 3.3 21.8 7.1 50.2 6.3 
SE 5.0 8.8 9.8 0.3 6.7 0.8 5.3 1.7 12.2 1.5 
Subjects Heat threshold Pain threshold Stim. int. (mJ/mm2Pain rating N1 lat. (ms) N1 amp. (μV) N2 lat. (ms) N2 amp. (μV) P2 lat. (ms) P2 amp. (μV) 
76 102 203 120 4.4 206 17.0 377 12.0 
51 178 203 122 4.4 172 7.9 218 4.7 
76 178 203 153 5.0 206 4.5 331 5.6 
51 76 203 126 7.3 167 5.4 239 4.4 
102 127 203 189 2.3 210 4.5 296 3.4 
76 102 178 106 7.4 184 9.6 241 5.8 
51 102 102 190 13.8 214 22.5 309 24.3 
76 178 203 185 1.6 240 3.3 380 7.2 
51 76 178 157 3.9 203 3.0 260 3.9 
10 76 102 254 168 3.9 200 4.3 331 7.2 
11 51 76 178 200 2.5 250 6.5 360 5.0 
12 102 127 178 161 4.4 198 5.3 267 14.8 
13 21 76 178 136 1.3 186 9.0 253 8.0 
14 76 102 127 170 5.4 190 14.0 281 21.0 
15 51 76 102 175 3.3 208 27.6 281 17.0 
16 76 102 127 161 4.2 185 4.6 240 6.8 
17 51 127 178 170 11.0 179 6.9 272 9.8 
AV 66 112 176 158 5.1 200 9.2 290 9.5 
SD 20.7 36.1 40.5 1.3 27.6 3.3 21.8 7.1 50.2 6.3 
SE 5.0 8.8 9.8 0.3 6.7 0.8 5.3 1.7 12.2 1.5 

Note that each value refers to the first LEP recording obtained in each subject. Stim. int., laser stimulation intensity (stimulator output); lat., latency; amp., amplitude; AV, average; SD, standard deviation; SE, standard error.

Experiment 1: Effect of Laser-PAS on MEP Amplitude

After Laser-PAS50, MEPs increased significantly in amplitude. The after-effects began 10 min after Laser-PAS50 ended and lasted <70 min. The MEP facilitation reached maximum at T4, T5, and T6 (30–50 min), with conditioned MEPs reaching 161% of baseline amplitude at T4, 178% at T5 and 165% at T6. Conversely, during Laser-PAS50, MEPs were initially inhibited and then returned to baseline values (reaching 74% of baseline amplitude at T0a, 72% at T0b and 94% at T0c).

In the whole group of 17 subjects, 1-way ANOVA showed a significant effect of the factor “Time” (F7,112 = 5.77; P < 0.01) and post hoc analysis showed that Laser-PAS50 increased MEP size significantly from T2 to T7 (P < 0.01 for all comparisons) (Fig. 2).

Figure 2.

Effect of Laser-PAS50 on motor-evoked potential (MEP) amplitudes (Experiment 1). Each point corresponds to the mean MEP amplitude recorded at baseline, during and 0–90 min after conditioning. Vertical bars denote SE. Note that during Laser-PAS50, MEPs decreased in amplitude whereas after Laser-PAS50 they increased and the increase was significant at 20–60 (T2–T7) but not at 70–90 min (T8–T10) after Laser-PAS50. MEPs from T0 to T7 were recorded in all 17 healthy subjects, whereas MEPs tested at T8, T9, and T10 refer to a subgroup of 5 subjects.

Figure 2.

Effect of Laser-PAS50 on motor-evoked potential (MEP) amplitudes (Experiment 1). Each point corresponds to the mean MEP amplitude recorded at baseline, during and 0–90 min after conditioning. Vertical bars denote SE. Note that during Laser-PAS50, MEPs decreased in amplitude whereas after Laser-PAS50 they increased and the increase was significant at 20–60 (T2–T7) but not at 70–90 min (T8–T10) after Laser-PAS50. MEPs from T0 to T7 were recorded in all 17 healthy subjects, whereas MEPs tested at T8, T9, and T10 refer to a subgroup of 5 subjects.

When we tested changes in MEP amplitudes in the subgroup of 5 subjects in whom MEP collection was prolonged to 90 min, 1-way ANOVA again showed that Laser-PAS50 increased MEPs significantly (significant effect of the factor “Time”; F10,40 = 20.13; P < 0.01). Post hoc tests specified that although Laser-PAS50 increased MEPs at T2–T7 (P < 0.05 for all comparisons), at T8, T9, and T10 it left them unchanged (P > 0.05 at all time points) (Fig. 2).

One-way ANOVA for MEPs collected during Laser-PAS50 showed a significant effect of the factor “Time” (F3,48 = 11.78; P < 0.01) and post hoc analysis demonstrated that Laser-PAS50 decreased MEP size significantly at T0a and T0b (P < 0.05 for all comparisons) but not at T0c (P > 0.05) (Fig. 2).

Experiment 2: Effect of Varying ISIs

In this experiment, we found that although Laser-PAS50 increased MEPs significantly, Laser-PAS0, Laser-PAS100 and Laser-PAS200 left MEPs unchanged. Two-way ANOVA showed a significant interaction between factors “ISI” and “Time” (F21,84 = 2.67; P < 0.01). Post hoc 1-way ANOVA showed that despite similar MEP amplitudes at T0 (Laser-PAS50: 0.9 ± 0.18 mV; Laser-PAS0: 0.7 ± 0.12 mV; Laser-PAS100: 0.92 ± 0.25 mV; Laser-PAS200: 0.79 ± 0.20 mV; P > 0.05 for all comparisons), Laser-PAS50 increased MEPs significantly as demonstrated by a significant effect of the factor “Time” (F7,28 = 5.07; P < 0.01), and did so at T2–T7 (P < 0.05 for all comparisons), whereas Laser-PAS0, Laser-PAS100 and Laser-PAS200 did not (Laser-PAS0: F7,28 = 1.74; P = 0.14; Laser-PAS100: F7,28 = 1.76; P = 0.13; Laser-PAS200: F7,28 = 1.37; P = 0.26) (Fig. 3).

Figure 3.

Effect of Laser-PAS delivered at laser-evoked potential (LEP) N1 + 0, 50, 100 and 200 ms ISI on motor-evoked potential (MEP) amplitudes (Experiment 2). Each point corresponds to the mean MEP amplitude recorded at baseline and 0–60 min after conditioning. Vertical bars denote SE. Note the significant increase in MEP amplitudes at 20–60 min (T2–T7) after Laser-PAS at LEP N1 + 50 but not at 0, 100 and 200 ms ISIs.

Figure 3.

Effect of Laser-PAS delivered at laser-evoked potential (LEP) N1 + 0, 50, 100 and 200 ms ISI on motor-evoked potential (MEP) amplitudes (Experiment 2). Each point corresponds to the mean MEP amplitude recorded at baseline and 0–60 min after conditioning. Vertical bars denote SE. Note the significant increase in MEP amplitudes at 20–60 min (T2–T7) after Laser-PAS at LEP N1 + 50 but not at 0, 100 and 200 ms ISIs.

Experiment 3: Topographic Specificity

ANOVA provided evidence that the Laser-PAS50-induced changes in MEP amplitudes were topographically restricted to the ADM muscle and did not spread to non-target muscles. Two-way ANOVA showed a significant interaction between factors “Muscle” and “Time” (F7,28 = 5.85; P < 0.01). Post hoc 1-way ANOVA showed that despite similar MEP amplitudes at T0 (ADM: 0.98 ± 0.19 mV; APB: 0.97 ± 0.16 mV; P > 0.05), Laser-PAS50 increased MEPs recorded from ADM muscle (F7,28 = 8.35; P < 0.01) and did so at T2–T7 (P < 0.05 at all time points), whereas MEPs from APB remained invariably unchanged (F7,28 = 1.87; P = 0.11) (Fig. 4).

Figure 4.

Effect of Laser-PAS50 on motor-evoked potentials (MEPs) elicited from abductor digiti minimi (ADM) and abductor pollicis brevis (APB) muscles (Experiment 3). Each point corresponds to the mean MEP amplitude recorded at baseline and 0–60 min after conditioning. Vertical bars denote SE. Note the significant increase in MEP amplitudes at 20–60 min (T2–T7) recorded from ADM but not from APB muscle.

Figure 4.

Effect of Laser-PAS50 on motor-evoked potentials (MEPs) elicited from abductor digiti minimi (ADM) and abductor pollicis brevis (APB) muscles (Experiment 3). Each point corresponds to the mean MEP amplitude recorded at baseline and 0–60 min after conditioning. Vertical bars denote SE. Note the significant increase in MEP amplitudes at 20–60 min (T2–T7) recorded from ADM but not from APB muscle.

Experiment 4: Effect of Repetitive Laser Pulses on MEP Amplitude

ANOVA showed that MEP amplitudes differed after Laser-PAS50 and 0.1 Hz laser. Two-way ANOVA showed a significant interaction between factors “Stimulation” and “Time” (F7,28 = 4.36; P < 0.01). Post hoc 1-way ANOVA showed that although MEP amplitudes at T0 were comparable (Laser-PAS50: 1.05 ± 0.16 mV; 0.1 Hz-laser: 0.9 ± 0.28 mV; P > 0.05), Laser-PAS50 increased MEPs (F7,28 = 4.63; P < 0.01) and did so at T2–T7 (P < 0.05 at all time points), whereas 0.1 Hz-laser stimulation did not (F7,28 = 1.2; P = 0.3) (Fig. 5).

Figure 5.

Effect of Laser-PAS50 and 0.1 Hz laser stimulation on motor-evoked potential (MEP) amplitudes (Experiment 4). Each point corresponds to the mean MEP amplitude recorded at baseline and 0–60 min after conditioning. Vertical bars denote SE. Note the significant increase in MEP amplitudes at 20–60 min (T2–T7) after Laser-PAS50 but not after 0.1 Hz laser stimulation.

Figure 5.

Effect of Laser-PAS50 and 0.1 Hz laser stimulation on motor-evoked potential (MEP) amplitudes (Experiment 4). Each point corresponds to the mean MEP amplitude recorded at baseline and 0–60 min after conditioning. Vertical bars denote SE. Note the significant increase in MEP amplitudes at 20–60 min (T2–T7) after Laser-PAS50 but not after 0.1 Hz laser stimulation.

Experiment 5: Effect of rTMS on MEP Amplitude

In this experiment, ANOVA showed that Laser-PAS50 and 0.1 Hz-rTMS changed MEP amplitudes in different ways. Two-way ANOVA showed a significant interaction between factors “Stimulation” and “Time” (F7,28 = 4.75; P < 0.01). Post hoc 1-way ANOVA showed that despite similar MEP amplitudes at T0 (Laser-PAS50: 0.92 ± 0.15 mV; 0.1 Hz-rTMS : 0.76 ± 0.28 mV; P > 0.05), Laser-PAS50 increased MEPs significantly (F7,28 = 14.77; P < 0.01) and did so at T2–T7 (P < 0.05 at all time points), whereas 0.1 Hz rTMS did not (F7,28 = 1.27; P = 0.3) (Fig. 6).

Figure 6.

Effect of Laser-PAS50 and 0.1 Hz-rTMS on motor-evoked potential (MEP) amplitudes (Experiment 5). Each point corresponds to the mean amplitude of MEPs recorded at baseline and 0–60 min after conditioning. Vertical bars denote SE. Note the significant increase in MEP amplitudes at 20–60 min (T2–T7) after Laser-PAS50 but not after 0.1 Hz-rTMS.

Figure 6.

Effect of Laser-PAS50 and 0.1 Hz-rTMS on motor-evoked potential (MEP) amplitudes (Experiment 5). Each point corresponds to the mean amplitude of MEPs recorded at baseline and 0–60 min after conditioning. Vertical bars denote SE. Note the significant increase in MEP amplitudes at 20–60 min (T2–T7) after Laser-PAS50 but not after 0.1 Hz-rTMS.

Experiment 6: Cortical versus Subcortical Mechanisms

ANOVA showed that although after Laser-PAS50, MEPs elicited by TMS increased significantly in amplitude, those evoked by TES did not. Two-way ANOVA showed a significant interaction between factors “Cortical versus subcortical activation” and “Time” (F7,28 = 2.83; P = 0.02). Post hoc 1-way ANOVA showed that despite similar MEP amplitudes at T0 (TMS: 0.73 ± 0.20 mV; TES: 0.78 ± 0.13 mV; P > 0.05), Laser-PAS50 increased MEPs elicited by TMS (F7,28 = 6.18; P < 0.01) and did so at T2–T7 (P < 0.05 at all time points), whereas MEPs evoked by TES remained unchanged at all time points (F7,28 = 0.85; P = 0.56) (Fig. 7).

Figure 7.

Effect of Laser-PAS50 on MEPs elicited by TMS and TES (Experiment 6). Each point corresponds to the mean amplitude of MEPs recorded at baseline and 0–60 min after conditioning. Vertical bars denote SE. Note the significant increase in MEP amplitudes at 20–60 min (T2–T7) elicited by TMS but not in those elicited by TES.

Figure 7.

Effect of Laser-PAS50 on MEPs elicited by TMS and TES (Experiment 6). Each point corresponds to the mean amplitude of MEPs recorded at baseline and 0–60 min after conditioning. Vertical bars denote SE. Note the significant increase in MEP amplitudes at 20–60 min (T2–T7) elicited by TMS but not in those elicited by TES.

Experiment 7: Effect of Laser-PAS with Laser Pulses Given Ipsilaterally to M1

In this experiment, ANOVA showed that Laser-PAS50, induced changes in MEP amplitudes only when laser pulses were given contralaterally to the stimulated M1. Two-way ANOVA showed a significant interaction between factors “Stimulation” and “Time” (F7,28 = 3.08; P = 0.02). Post hoc 1-way ANOVA showed that although comparable MEP amplitudes at T0 (Laser-PAS50: 1.05 ± 0.16 mV; Laser-PAS50 with ipsilateral laser stimuli: 0.85 ± 0.16 mV; P > 0.05), Laser-PAS50 increased MEPs (F7,28 = 14.77; P < 0.01) and did so at T2–T7 (P < 0.05 at all time points), whereas Laser-PAS50 with ipsilateral laser stimuli did not (F7,28 = 1.32; P = 0.28) (Fig. 8).

Figure 8.

Effect of Laser-PAS50 on MEPs with laser stimuli given ipsilaterally and contralaterally to the stimulated M1 (Experiment 6). Each point corresponds to the mean amplitude of MEPs recorded at baseline and 0–60 min after conditioning. Vertical bars denote SE. Note the significant increase in MEP amplitudes at 20–60 min (T2–T7) elicited by Laser-PAS50 implying laser stimuli given contralaterally but not ipsilaterally to the stimulated M1.

Figure 8.

Effect of Laser-PAS50 on MEPs with laser stimuli given ipsilaterally and contralaterally to the stimulated M1 (Experiment 6). Each point corresponds to the mean amplitude of MEPs recorded at baseline and 0–60 min after conditioning. Vertical bars denote SE. Note the significant increase in MEP amplitudes at 20–60 min (T2–T7) elicited by Laser-PAS50 implying laser stimuli given contralaterally but not ipsilaterally to the stimulated M1.

Experiment 8: Effect of a Drug Acting on the NMDA Receptor

This experiment demonstrated that the Laser-PAS50-induced changes in MEP amplitudes were abolished in subjects receiving memantine but not in those receiving placebo. Two-way ANOVA showed a significant interaction between factors “Drug” and “Time” (F7,49 = 3.57; P < 0.01). Post hoc 1-way ANOVA showed that despite similar MEP amplitudes at T0 (memantine: 0.85 ± 0.08 mV; placebo: 0.80 ± 0.07 mV; P > 0.05), after Laser-PAS50 in subjects receiving memantine MEP amplitudes remained unchanged (F7,49 = 0.44; P = 0.87), whereas in subjects receiving placebo they increased significantly (F7,49 = 8.54; P < 0.01) and did so at T2–T7 (P < 0.05 at all time points) (Fig. 9).

Figure 9.

Effect of Laser-PAS50 on MEP amplitudes in subjects receiving memantine or α-lipoic acid (Experiment 8). Each point corresponds to the mean amplitude of MEPs recorded at baseline and 0–60 min after conditioning. Vertical bars denote SE. Note the significant increase in MEP amplitudes at 20–60min (T2–T7) in subjects receiving α-lipoic acid but not in those receiving memantine.

Figure 9.

Effect of Laser-PAS50 on MEP amplitudes in subjects receiving memantine or α-lipoic acid (Experiment 8). Each point corresponds to the mean amplitude of MEPs recorded at baseline and 0–60 min after conditioning. Vertical bars denote SE. Note the significant increase in MEP amplitudes at 20–60min (T2–T7) in subjects receiving α-lipoic acid but not in those receiving memantine.

One-way ANOVA showed that RMT remained unchanged over time in all experiments. Similarly, in Experiment 6, RMTelectric also remained unchanged from T0 to T7. In addition, RMT and the intensity required to evoke 1 mV MEPs at T0 were similar in all experimental sessions. Finally, the Kruskal–Wallis Test showed comparable laser perceptive thresholds and pain ratings for each subject in all the experimental sessions.

Spearman correlation test showed no correlation in any subject involved in the study between Laser-PAS50-induced changes in MEP amplitude, laser perceptive threshold and pain rating, RMT and the intensity used for evoking 1 mV MEPs at T0.

Discussion

In the healthy subjects we tested, the new PAS protocol we designed by combining painful skin laser pulses that selectively activate the nociceptive system and TMS, induced marked changes in M1 plasticity as measured by after-effects on MEP amplitudes. The MEP changes depended on specific ISIs (50 ms) (Laser-PAS50), and remained topographically restricted to the target ADM muscle. Unlike Laser-PAS50, repetitive laser pulses given alone and rTMS in a single train both failed to elicit changes in MEP amplitudes hence they left M1 excitability unchanged. Our finding that TMS-induced MEPs increased whereas TES-induced MEPs did not suggests that Laser-PAS50 induces changes in cortical rather than cortico-spinal axon excitability. Laser-PAS50 using laser pulses given ipsilaterally to the stimulated M1 failed to elicit changes in MEP sizes. Finally, memantine abolished the Laser-PAS50-induced after-effects, whereas α-lipoic acid did not suggesting that Laser-PAS50 activates the NMDA receptor. Overall these findings provide new important information on the physiological mechanisms underlying human pain-motor integration.

We took several precautions to ensure that our Laser-PAS study provided reliable results. For example, by randomly studying subjects in separate sessions and allowing at least 1 week to elapse between sessions we can confidently exclude interference between experimental sessions due to repeated Laser-PAS. Ample evidence shows that repeatedly applying plasticity study protocols significantly alters outcome measurements and does so through homeostatic or non-homeostatic metaplasticity mechanisms (Bienenstock et al. 1982; Abraham and Bear 1996; Davis 2006; Muller et al. 2007; Ziemann et al. 2008). The similar RMT and intensity for baseline MEPs in each subject also excludes between-session differences in baseline cortical excitability. Finally, because each subject had a similar laser perceptive threshold and gave similar pain ratings, received Laser-PAS at similar intensities, and also had similar LEP latencies and amplitudes among sessions, we can reasonably exclude differences related to repeated exposure to experimental pain.

Although the MEP increase seen when we paired repetitive laser pulses and TMS at the 50 ms ISI (Laser-PAS50) could in theory depend partly on changes in subjects' attention due to repetitive pain exposure and pain expectation (Koyama et al. 2005; Legrain et al. 2011; Mouraux et al. 2011) attentional differences seem unlikely given that Laser-PAS induced no MEP amplitude changes at the other ISIs tested. The observation that only Laser-PAS at the 50 ms ISI elicited after-effects also excludes the possibility that repeatedly applying TMS required for post-intervention MEP recordings (batches of 20 MEPs for 60 min in all subjects and experiments) might have somehow affected M1 responses to Laser-PAS. Laser-PAS probably induced after-effects only when delivered at the N1 + 50 ms ISI because this ISI in a PAS protocol elicits the maximal interaction between LEP-TMS. Hence like the MEP changes induced by PAS, Laser-PAS50-induced changes in MEP amplitudes depend crucially on the associative stimulation protocol delivered at the specific ISIs tested (Stefan et al. 2000; Ziemann et al. 2008).

When we investigated whether the Laser-PAS50–induced after-effects remain restricted to the target ADM muscle rather than spread to non-target muscles, we found that after Laser-PAS50 MEPs in the ADM increased, whereas in the APB muscle they did not. Like the original PAS protocol (Stefan et al. 2000), Laser-PAS50 elicits changes in MEP amplitudes only in the target muscle, suggesting that Laser-PAS50 elicits specific topographical changes.

We interpret our observation that Laser-PAS50 induced a MEP amplitude increase whereas repetitive laser pulses and rTMS given alone at 0.1 Hz did not, as confirming that the Laser-PAS50-induced after-effects reflect changes related to associative stimulation rather than simply reflecting changes elicited by repetitive laser stimuli or rTMS. Our data also agree with previous studies showing that 0.1 Hz-rTMS elicits no changes in MEP amplitudes (Chen et al. 1997; Cincotta et al. 2003; Fitzgerald et al. 2006).

Given that anodal TES, unlike biphasic PA–AP TMS, elicits MEPs predominantly by directly exciting cortico-spinal axons (Day et al. 1987; Petersen et al. 2003; Di Lazzaro et al. 2004), our study provides evidence showing that Laser-PAS50–induced after-effects reflect cortical mechanisms. In humans, direct recordings from the cervical spinal epidural space of descending cortico-spinal activity evoked by anodal TES and biphasic PA–AP TMS have demonstrated that anodal TES preferentially recruits D-waves and at higher intensities also I1-waves, whereas biphasic PA–AP TMS preferentially recruits I3-waves together with a “proximal D-wave” (sDi Lazzaro et al. 2001, 2004). Hence, after Laser-PAS50, the concurrent increased PA–AP TMS-induced and unchanged TES-induced MEPs presumably depended on excitability changes in M1 interneurons responsible for later I wave inputs to cortico-spinal neurons (Di Lazzaro et al. 2001, 2004; Petersen et al. 2003).

Precisely which LEP component, the LEP N1 or the central N2–P2 (Spiegel et al. 1996; Bromm and Lorenz 1998; Garcia-Larrea et al. 2003; Cruccu et al. 2008), is responsible for the Laser-PAS50-induced after-effects we observed is difficult to state. Our finding that Laser-PAS50 using laser pulses given ipsilaterally to the stimulated M1 failed to elicit after-effects strongly implicates the LEP N1 component as the predominant source of afferent inputs to M1 during Laser-PAS50.

Further important information on the mechanisms underlying Laser-PAS50-induced after-effects comes from our pharmacological study in subjects receiving an NMDA antagonist (memantine) or placebo (α-lipoic acid). Although memantine abolished the Laser-PAS50–induced after-effects whereas α-lipoic acid did not, given that neither memantine nor α-lipoic acid influenced RMT, the intensity for eliciting baseline MEPs (Schwenkreis et al. 1999; Huang et al. 2007), laser perceptive thresholds or pain ratings, we consider it highly unlikely that Laser-PAS50 failed to induce after-effects in subjects receiving memantine owing to drug-induced sedation or inattention. Conversely, we suggest that memantine abolished the Laser-PAS50-induced after-effects by inducing changes in NMDA-dependent glutamatergic transmission. Our findings are in line with previous studies showing that the NMDA antagonists memantine and dextromethorphan block the after-effects induced by various rTMS protocols (Stefan et al. 2000, 2002; Huang et al. 2005, 2007).

The new Laser-PAS technique resembles the original PAS protocol in several ways. For example, PAS, Laser-PAS50 drives neurophysiological mechanisms characterized by timing-dependent associativity, topographical specificity and M1 NMDA-dependent glutamatergic transmission (Stefan et al. 2000; Wolters et al. 2003). Despite the similarities, the 2 protocols also differ. Laser-PAS implies laser-induced peripheral Aδ nociceptor activation and afferent volleys conducted along small-myelinated fibres to SII, ACC and insula (Treede 1995; Garcia-Larrea et al. 2003). Conversely, PAS reflects peripheral nerve electric activation and afferent inputs conducted by large-myelinated fibres to the primary somatosensory area. The lack of LTD-like plasticity after Laser-PAS at the ISIs we studied might depend partly on signal dispersion in the afferent volleys travelling along the nociceptive fibres (Cruccu et al. 2003). Whether Laser-PAS induces LTD at ISIs other than those here investigated remains an open question for further research.

Our findings obtained with Laser-PAS50 may help us to explain plasticity mechanisms operative in human M1. Like the original PAS protocol, Laser-PAS50 elicits NMDA-related spike timing-dependent plasticity (STDP) (Stefan et al. 2000; Ziemann et al. 2008). Cortical STDP is thought to depend on coincidence detection of appropriately timed excitatory post-synaptic potentials in apical dendrites (layer 2/3) and back-propagating action potentials from layer 5 neurons (Stuart and Sakmann 1994; Markram et al. 1997; Song et al. 2000; Dan and Poo 2004, 2006; Caporale and Dan 2008). Given that TMS is thought to activate layer 5 cortico-spinal neurons indirectly by activating layers 2 and 3 pyramidal axons (Di Lazzaro et al. 2004, 2011), and that projections coming from remote pain-related cortical regions reach M1 in the 2 and 3 cortical layers (Asanuma and Keller 1991; Kaneko et al. 1994; Dum and Strick 2002, 2005; Dum et al. 2009), we conjecture that Laser-PAS50 probably elicited NMDA-dependent STDP in M1 cortical layers 2 and 3. Finally, insofar as STDP mechanisms intervene in experience-dependent learning depending on the temporal sequence and interval between pre- and postsynaptic activity (Cruikshank and Weinberger 1996; Buonomano and Merzenich 1998; Song et al. 2000; Bi and Poo 2001; Dan and Poo 2004, 2006; Caporale and Dan 2008; Feldman 2009), we suggest that the mechanisms underlying Laser-PAS50-induced NMDA-dependent STDP might be engaged in pain-motor integration processes.

Given that nociceptive inputs influence the motor system through functional connectivity between brain areas, the plasticity changes induced by Laser-PAS50 in healthy subjects might reflect functional connectivity between pain and cortical motor areas. Neurophysiological and neuroimaging studies increasingly identify a cortical network (including the ACC, insula, and SII) activated by various types of experimental pain as the “pain matrix”. The pain matrix is thought to be responsible for cortical elaboration and conscious experience of pain (Jones et al. 1992; Ploghaus et al. 1999; Treede et al. 1999; Peyron et al. 2000; Garcia-Larrea et al. 2003; Apkarian et al. 2005; Iannetti et al. 2005; Tracey and Mantyh 2007). The pain matrix may also include brain regions primarily involved in motor function including the supplementary motor area and M1 suggesting functional connectivity between the pain matrix and cortical motor areas (Peyron et al. 2000; Koyama et al. 2005; Tracey and Mantyh 2007; Iannetti and Mouraux 2010). Our findings might therefore imply that Laser-PAS50 activates functional connectivity between the pain matrix and M1. Given that both the ACC and SII connect closely to cortical motor areas and participate in motor control and motor learning processes, we find it difficult to clarify which brain region specifically contributed to our findings (Mori et al. 1989; He et al. 1995; Picard and Strick 1996; Forss and Jousmaki 1998; Paus 2001; Dum and Strick 2002, 2005; Disbrow et al. 2003; Dum et al. 2009). The lateralized LEP N1 component predominantly arises from SII and posterior insula (operculoinsular region), whereas the central N2–P2 component reflects ACC and bilateral insular activation (Spiegel et al. 1996; Bromm and Lorenz 1998; Garcia-Larrea et al. 2003; Cruccu et al. 2008). Hence, the observation that Laser-PAS50 failed to induce after-effects when we applied laser pulses ipsilaterally to the stimulated M1 strongly suggests that the after-effects here reported arose mainly through functional connectivity between SII, posterior insula, and M1.

When we tested possible MEP changes during Laser-PAS50, we found that when Laser-PAS50 began, MEPs decreased and when it ended returned to baseline values. Hence, a further comment concerns why Laser-PAS50 elicits STDP whereas a single laser-TMS–paired pulse induces M1 inhibition (Valeriani et al. 1999). Other PAS protocols have yielded similar findings (Stefan et al. 2000, Rizzo et al. 2009). PAS at 25 ms ISI induces MEP facilitation (Stefan et al. 2000), whereas a single median nerve electric stimulation preceding TMS at similar ISIs inhibits MEPs owing to short-afferent inhibition (Tokimura et al. 2000). Similarly, cortico-cortical PAS (ccPAS), consisting in repetitive test pulses over M1 and conditioning pulses over the contralateral M1 at 8 ms ISI, increases MEP size (Rizzo et al. 2009), whereas paired pulses applied at similar ISIs, owing to interhemispheric inhibition (Ferbert et al. 1992; Gerloff et al. 1998), inhibit MEP size. Hence, repeatedly applying specific inhibitory paired-pulse protocols in a PAS design may turn M1 excitability from inhibition to facilitation. In none of the subjects studied in Experiment 1 did we find a correlation between the magnitude of MEP inhibition during Laser-PAS50 and the magnitude of MEP facilitation after Laser-PAS50. We therefore suggest that the physiological mechanisms underlying MEP inhibition during Laser-PAS50 bear no relation to those responsible for Laser-PAS50-induced LTP-like plasticity. The MEP inhibition observed during Laser-PAS50 agrees with the previously reported LEP-induced M1 inhibition interpreted with the pain adaptation model which predicts that pain may drive cortical motor areas to decrease their overall activity to promote protective pain-induced spinal reflexes (Lund et al. 1991; Valeriani et al. 1999, 2001; Murray and Peck 2007; Legrain et al. 2011). Although this explanation might hold true when subjects receive few painful stimuli, under experimental conditions entailing repetitive painful stimulation cortical motor areas might increase rather than decrease their overall activity to promote cortical strategies for eliminating the painful stimulation and preventing further injury.

In conclusion, our new Laser-PAS50 method provides new insight into the physiological role of pain-motor integration processes in humans. The Laser-PAS50 protocol might also help in verifying whether possible abnormalities in pain-motor integration processes contribute to the pathophysiology of chronic pain (Karl et al. 2001; Reilly et al. 2006; MacIver et al. 2008). Finally, Laser-PAS50 might also open up novel therapeutic rTMS approaches for chronic pain (Attal et al. 2006; Cruccu et al. 2007).

Notes

Conflict of Interest: None declared.

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