Brain Stimulation 7 (2014) 113e121

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Increased Transcranial Direct Current Stimulation After Effects During Concurrent Peripheral Electrical Nerve Stimulation Vincenzo Rizzo a, Carmen Terranova a, Domenica Crupi d, Antonino Sant’angelo b, Paolo Girlanda a, Angelo Quartarone a, c, * a

Department of Neurosciences, Psychiatry and Anaesthesiology, University of Messina, Italy Fondazione Istituto “San Raffaele - G. Giglio,” Unit for Severe Acquired Brain Injuries, Rehabilitation Department, Cefalù, Italy c Department of Neurology, NYU School of Medicine, New York, NY, USA d Regional Epilepsy Centre “Bianchi-Melacrino-Morelli” Hospital, Reggio Calabria, Italy b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 May 2013 Received in revised form 25 September 2013 Accepted 13 October 2013

In this study we tested the hypothesis whether a lasting change in the excitability of cortical output circuits can be obtained in healthy humans by combining a peripheral nerve stimulation during a concomitant depolarization and/or hyperpolarization of motor cortex. To reach this aim we combined two different neurophysiological techniques each of them able to induce a lasting increase of cortical excitability by them self: namely median nerve repetitive electrical stimulation (rEPNS) and transcranial direct current stimulation (tDCS). Ten normal young volunteers were enrolled in the present study. All subjects underwent five different protocols of stimulation: (1, 2) tDCS alone (anodal or cathodal); (3) Sham tDCS plus rEPNS; (4, 5) anodal or cathodal tDCS plus rEPNS. The baseline MEP amplitude from abductor pollicis brevis (APB) and flexor carpi radialis (FCR) muscle, the FCR H-reflex were compared with that obtained immediately after and 10, 20, 30, 60 min after the stimulation protocol. Anodal tDCS alone induced a significant transient increase of MEP amplitude immediately after the end of stimulation while anodal tDCS þ rEPNS determined MEP changes which persisted for up 60 min. Cathodal tDCS alone induced a significant reduction of MEP amplitude immediately after the end of stimulation while cathodal tDCS þ rEPNS prolonged the effects for up to 60 min. Sham tDCS þ rEPNS did not induce significant changes in corticospinal excitability. Anodal or cathodal tDCS þ rEPNS and sham tDCS þ rEPNS caused a lasting facilitation of H-reflex. These findings suggest that by providing afferent input to the motor cortex while its excitability level is increased or decreased by tDCS may be a highly effective means for inducing an enduring bi-directional plasticity. The mechanism of this protocol may be complex, involving either cortical and spinal after effects. Ó 2014 Elsevier Inc. All rights reserved.

Keywords: Plasticity Motor cortex Transcranial magnetic stimulation Transcranial direct current stimulation Peripheral electrical current stimulation

Introduction Plasticity is the property of the central nervous system to change the effectiveness of transmission in neural circuits in response to various environmental changes, injuries or in association with skill acquisition. Long-term potentiation (LTP) has been considered one of the candidate mechanisms for cortical plasticity and is characterized by a persistent enhancement of synaptic strength [1]. LTP may be induced in animal models by using both ‘high-frequency stimulation’ (HFS) protocols (i.e., ‘theta-burst stimulation’) and ‘paired associative stimulation’ (PAS) protocols. The former approach delivers strong tetanizing pre-synaptic stimuli along a single pathway spaced at a frequency that mimics a spontaneous * Corresponding author. Clinica Neurologica 2, Policlinico Universitario, 98125 Messina, Italy. Tel.: þ39 90 2212791; fax: þ39 90 2212789. E-mail address: [email protected] (A. Quartarone). 1935-861X/$ e see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brs.2013.10.002

5e7 Hz neural rhythm, the theta wave. Tetani of this sort can be modeled on the human side using transcranial magnetic stimulation (TMS) and when applied repetitively can induce long-lasting changes in motor cortical output [2]. The latter approach, called PAS, couples two temporally-related inputs that reach simultaneously the post-synaptic cell [3,4]. More in detail, PAS occurs when an input to a post-synaptic cell is synchronous to (i) another input to the same cell or concomitant to (ii) a post-synaptic depolarization provided by intracellular injected depolarizing currents in the post-synaptic cell [5]. Pairing of this sort, as indicated in (i), can potentially be modeled in humans by combining low or high frequency TMS to the cortex whilst simultaneously stimulating a peripheral nerve [6,7]. In particular PAS combines an electrical stimulus of the median nerve with a TMS pulse to the motor cortex. If the stimuli are timed with an interstimulus interval (ISI) such that the afferent input

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reaches the motor cortex just before the TMS, then repeated pairings lead to an enduring facilitation of excitability [6,7]. Vice versa if the order of stimulation was reversed (i.e., the afferent input reaches the motor cortex just after the TMS), it produces instead a reduction of cortical excitability [8]. However, to our knowledge there is no report of human PAS paradigms similar to that of condition (ii) described in animal models where an input to a post-synaptic cell is synchronous to a concomitant post-synaptic depolarization. Hence in the present paper, we tested the hypothesis whether a lasting change in the excitability of cortical output circuits can be obtained in healthy humans by combining a peripheral nerve stimulation during a concomitant depolarization and/or hyperpolarization of motor cortex. To reach this aim we combined two different neurophysiological techniques each of them is able to induce a lasting increase of cortical excitability by themselves: median nerve repetitive electrical stimulation (rEPNS) [9] and transcranial direct current stimulation (tDCS) [10]. In human subjects tDCS can modify motor cortex excitability dependent on stimulation polarity, intensity and duration [10]. The cellular mechanisms of tDCS are not completely understood. A fraction of the current is thought to enter the brain and to change the polarization of neurons in the vicinity of the electrodes [11]. Our hypothesis is that providing median nerve rEPNS reaches the somatosensory and motor cortex at a time when the membrane potential of its related pyramidal neuron is biased by tDCS is sufficient to induce enduring changes of motor cortex excitability in a more effective way of that induced by tDCS alone. Materials and methods Participants Ten right-handed healthy volunteers (5 female and 5 male), aged 26e38 years (mean age 30  3 years), participated in the study. The experiment was approved by the local Ethics Committee. All subjects gave their written informed consent for the experiments. Subjects were seated in a comfortable reclining chair during the experiment. A pillow supported both arms. They were asked to completely relax and to look straight ahead. Main experiment Figure 1 illustrates the timeline of the experimental procedures. The main experiment was designed to assess the conditioning effects of rEPNS þ tDCS protocol to the left M1 on various measures

of motor cortical excitability. All subjects were studied during each of five experimental paradigms. Motor evoked potentials (MEP) were recorded from the abductor pollicis brevis (APB) muscle at six different blocks: immediately before (baseline) and after conditioning protocol to the left M1 (T0) as well as 10, 20, 30 and 60 min (T10, T20, T30 and T60) after. In each block, we first measured motor threshold (MT) at rest and during tonic contraction. Threshold measurements were followed by measurements of corticospinal excitability. Conditioning protocol The conditioning protocol included five main experimental sessions separated by approximately one week, in which, participants received: 1) anodal tDCS þ rEPNS; 2) cathodal tDCS þ rEPNS; 3) anodal tDCS; 4) cathodal tDCS; 5) sham tDCS þ rEPNS. Participants were blinded for the type of experimental sessions. The order of experimental sessions was pseudorandomized. For tDCS a constant direct current of 1 mA intensity was applied through saline-soaked sponge electrodes (surface 35 cm2). For cathodal stimulation the cathode was placed above the motor cortical representational field of the right abductor pollicis brevis (APB) muscle, as revealed by TMS, and the anode above the contralateral orbita. For anodal stimulation the montage was reversed. Cathodal and anodal tDCS were delivered through a battery-driven constantcurrent stimulator (DC stimulator, Rolf Schneider electronic, 37130 Gleichen, Germany) for a period of 5 min. Constant current flow was monitored by a voltmeter. For sham tDCS, the stimulator was switched on for 5 s and then turned off for the remaining 5 min. rEPNS consisted of 1500 stimuli which were continuously delivered to the left median nerve at a rate of 5 Hz for 5 min. A square wave pulse was applied to the right median nerve at the wrist through a bipolar electrode (Digitimer D-160 stimulator; Digitimer Ltd, Welwyn Garden City, Herts, UK). The cathode was located proximally and the pulse width was 500 ms. The intensity of the electrical stimulus was set at twice the sensory threshold. Measures of cortical excitability The effects of conditioning protocol on motor cortical excitability were probed using TMS pulses with a monophasic pulse configuration. Single pulse was given to the left M1 using a standard figure-of-eight coil connected with a single high-power Magstim 200 stimulator. The coil had an external loop diameter of 9 cm. The center of the coil was located over the ‘motor hot spot’ for stimulation of the contralateral APB muscle and the handle of the coil

Figure 1. Experimental procedures. Subjects underwent five main experimental sessions separated by approximately one week. In each session, participants received: 1) anodal tDCS þ rEPNS; 2) cathodal tDCS þ rEPNS; 3) anodal tDCS; 4) cathodal tDCS; 5) sham tDCS þ rEPNS. Motor evoked potentials (MEPs) were recorded from the right abductor pollicis brevis (APB). Electrophysiological measurements were performed before and up to 60 min after the conditioning at five time points (immediately ¼ T0; 10 min ¼ T10; 20 min ¼ T20, 30 min ¼ T30; 60 min ¼ T60). In each block of measurement, we assessed resting motor threshold (RMT), active motor threshold (AMT), mean MEP amplitudes.

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pointed 45 postero-laterally. The monophasic magnetic stimulus had a rise time of approximately 100 ms, decaying back to zero over approximately 0.8 ms. The coil current during the rising phase of the magnetic field flowed toward the handle. Thus, the induced current in the cortex flowed in a posterioreanterior direction. Threshold measurements Resting MT was defined as the minimum intensity that evoked a peak-to-peak MEP of 50 mV in at least 5 out of 10 consecutive trials in the relaxed APB muscle. Active MT was defined as the minimum intensity that elicited a reproducible MEP of at least 200 mV in the tonically contracting APB muscle in at least 5 out of 10 consecutive trials. Participants maintained a force level of approximately 10e15% of maximum force during measurements of the active MT. Corticospinal excitability The MEP peak-to-peak amplitude was taken as a measure of corticospinal excitability. To assess mean peak-to-peak MEP amplitudes at rest, 20 monophasic magnetic stimuli were given to the motor hot spot of the APB muscle at a rate of 0.1 Hz. Stimulus intensity was set at a stimulator output that induced MEPs of 0.5e1 mV (w115e125% of resting MT) in the right APB muscle. This intensity was defined prior to baseline measurements and was kept constant throughout the experiment. Trials in which the APB muscle was not completely relaxed were discarded from analysis. Additional experiments In addition to the main experiment, we performed additional experiments to further characterize the conditioning effects of the rEPNS þ tDCS protocol. The experiments were carried out in subgroups of the volunteers who had participated in the main experiment.

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experimental sessions were given at least 1 week apart and were pseudorandomized. CSP was tested during slight tonic contraction of the right APB muscle at approximately 10e15% of maximum force level. Audiovisual feedback of ongoing EMG activity was provided to ensure a constant force level. Stimulus intensity was identical to the stimulus intensity used for the test stimulus during paired-pulse TMS (w115e125% of resting MT). The duration of the CSP is a marker for the excitability of long-lasting (presumably GABABergic) intracortical inhibition [13,14]. For CSP measurements, EMG traces were rectified but not averaged. The duration of the CSP was measured in each trial and defined as the time from the onset of the MEP to reappearance of sustained EMG activity [15]. Short afferent inhibition (SAI) and long afferent inhibition (LAI) In 5 subjects (3 female and 2 male; mean age: 28 years; age range: 26e38 years), we evaluated SAI and LAI immediately before (baseline) and after anodal or cathodal tDCS þ rEPNS to the left M1 (T0) as well as 10, 20 and 30 min (T10, T20 and T30) after conditioning protocol. The two experimental sessions were given at least 1 week apart and were pseudorandomized. SAI and LAI were assessed in the APB muscle using the techniques described respectively by Tokimura and Chen [16,17]. In brief, the intensity of the test TMS pulse was adjusted to produce a MEP of approximately 0.5e1 mV peak to peak. The conditioning pulse was an electrical stimulus to the median nerve at the wrist (cathode proximal) using a square wave pulse with a pulse width of 200 ms. The intensity was set just above the threshold for evoking a visible twitch of the thenar muscles (approximately three times perceptual threshold). The interstimulus interval was 25 ms for SAI and 200 ms for LAI. Twenty unconditioned and conditioned responses were randomly intermixed and the mean amplitude of the conditioned response expressed as a percentage of the test response alone.

Short-latency intracortical inhibition (SICI) and intracortical facilitation (ICF) In 8 subjects (4 male and 4 female; mean age: 30 years; age range: 26e38 years), we tested ICI and ICF immediately before (baseline) and after anodal or cathodal tDCS þ rEPNS to the left M1 (T0) as well as 10, 20, 30 and 60 min (T10, T20, T30 and T60) after conditioning protocol. The two experimental sessions were given at least 1 week apart and were pseudorandomized. SICI and ICF were determined according to the paired-pulse method described by Kujirai et al. [12]. The intensity of the conditioning stimulus was set at 80% of active MT. The intensity of the test stimulus was adjusted to elicit MEPs with peak-to-peak amplitudes of 0.5e1.0 mV at baseline (w115e125% of resting MT). Stimulus intensities were kept constant across the blocks of measurement. SICI and ICF were assessed at ISIs of 2 and 12 ms, respectively. Twenty trials were recorded for each ISI and randomly intermingled with twenty trials in which MEPs were elicited by the test stimulus alone. The peak-to-peak amplitude of the unconditioned MEP was taken as a measure of corticospinal excitability. Mean amplitude of the conditioned MEP was expressed as percentage of the amplitude of the unconditioned MEP. The relative change in MEP amplitude induced by the conditioning stimulus characterized the strength of SICI and ICF. Trials in which the APB muscle was not completely relaxed were discarded from analysis.

Spinal cord excitability In 5 subjects (2 female and 3 male; mean age: 29 years; age range: 26e38 years), we measured the flexor carpi radialis (FCR) Hreflex as previously described in literature [18] and MEP amplitude recorded from FCR. H-reflex and MEP amplitude were assessed immediately before (baseline) and after conditioning protocol to the left M1 (T0) as well as 10, 20 and 30 min (T10, T20 and T30) after. The conditioning protocol included the same five sessions of the main experiment separated by approximately one week. Percutaneous electrical stimulation of the median nerve (rectangular pulse of 1 ms duration, 0.33 Hz) was used to evoke an H-reflex in the FCR. A marked increase in the H-reflex during wrist flexion but not during pure pronation or finger flexion was used as a criterion indicating that the reflex originated mainly from FCR. The FCR H-reflex was evoked by stimulating the median nerve through a bipolar electrode placed 2 cm below the elbow in the medial side of the arm. Before each experiment, the maximal Hreflex response (Hmax) and the maximal M response (Mmax) were recorded. As the sensitivity of the H-reflex to facilitatory or inhibitory conditioning effects is known to depend on its size [19], the size of the FCR control H-reflexes was systematically adjusted to between 40% and 50% of Mmax.

Cortical silent period We measured cortical silent period (CSP) in 8 subjects (4 male and 4 female; mean age: 30 years; age range: 26e38 years) immediately before (baseline) and after anodal or cathodal tDCS þ rEPNS to the left M1 (T0) as well as 10, 20, 30 and 60 min (T10, T20, T30 and T60) after conditioning protocol. The two

EMG was recorded with AgeAgCl surface electrodes from the right APB and FCR muscles using a belly-tendon montage. The signal was amplified and bandpass filtered (20 Hze1 KHz) by a Digitimer D-150 amplifier (Digitimer Ltd., Welwyn Garden City, Herts, UK) and stored at a sampling rate of 10 KHz on a personal computer for off-line analysis (Signal Software, Cambridge

Data acquisition

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Figure 2. A) Effects of anodal tDCS þ rEPNS, anodal tDCS and sham tDCS þ rEPNS on MEP amplitude recorded from APB muscle. Anodal tDCS þ rEPNS induces an increase in MEP amplitude at each time (T0: P < 0.0001; T10: P < 0.0001; T20: P < 0.0001; T30: P < 0.0003 and T60: P < 0.0003) relative to baseline. Anodal tDCS alone induces an increase in MEP amplitude only at T0 (P < 0.0001) and T10 (P ¼ 0.02). Error bars indicate standard error of the mean. *P  0.05. B) Effects of cathodal tDCS þ rEPNS, cathodal tDCS and sham tDCS þ rEPNS on MEP amplitude recorded from APB muscle. Cathodal tDCS þ rEPNS induces a decrease in MEP amplitudes at each time (T0: P < 0.0001; T10: P < 0.0001; T20: P < 0.0001; T30: P < 0.0001 and T60: P < 0.0001) relative to baseline. Cathodal tDCS induces a depression in MEP amplitude at only at T0 (P < 0.0001). Error bars indicate standard error of the mean. *P  0.05.

Electronic Design, Cambridge, UK). During the experiments EMG activity was continuously monitored with visual (oscilloscope) and auditory (speakers) feedback to ensure either complete relaxation at rest or a constant level of EMG activity during tonic contraction. Statistical analysis The conditioning effects of different conditioning protocol on motor thresholds (resting and active MT), peak-to-peak MEP amplitude of responses to single-pulse TMS, duration of CSP, paired-pulse intracortical excitability (SICI and ICF), afferent inhibition (SAI and LAI) and spinal cord excitability (H-reflex) were evaluated in separate repeated-measures analyses of variance (rANOVA). For MEP amplitude and H-reflex, we computed a twoway repeated-measures ANOVA having time (6 levels: baseline, T0, T10, T20, T30, and T60) and conditioning (3 levels for anodal stimulation: anodal tDCS þ rEPNS vs anodal tDCS vs sham tDCS þ rEPNS and 3 levels for cathodal stimulation: cathodal tDCS þ rEPNS vs cathodal tDCS vs sham tDCS þ rEPNS) as withinsubject factor. For the evaluation of motor thresholds, the state of the muscle (relaxation vs contraction) was included as additional factor in the ANOVA. ISI was considered as additional factors in the ANOVAs testing for changes in paired-pulse measurements (i.e., SICI, ICF, SAI and LAI). The GreenhouseeGeisser method was used if

necessary to correct for non-sphericity. Conditional on a significant F-value, post hoc paired samples t tests were performed to explore the strength of main effects and the patterns of interaction between experimental factors. A P-value of 0.3). Anodal tDCS þ rEPNS caused a lasting facilitation of MEP amplitudes with respect to anodal tDCS alone (Fig. 2A). Post hoc t tests for anodal tDCS þ rEPNS revealed an increase in MEP amplitude at T0 (t1,9 ¼ 5.3, P < 0.0001), T10 (t1,9 ¼ 6.8, P < 0.0001), T20 (t1,9 ¼ 4.3, P < 0.0001), T30 (t1,9 ¼ 4.0, P < 0.0003) and T60 (t1,9 ¼ 3.9, P < 0.0003) relative to baseline. Instead, post hoc t tests for anodal tDCS revealed an increase in MEP amplitude at T0 (t1,9 ¼ 6.7, P < 0.0001) and T10 (t1,9 ¼ 2.7, P ¼ 0.02) alone. Moreover, cathodal tDCS þ rEPNS modified also mean peak-topeak MEP amplitude. The ANOVA showed an interaction between time and conditioning (F10,90 ¼ 13.5, P < 0.0001). Separate followup ANOVAs with time as a within-subject factor were performed to characterize time-dependent changes in MEP amplitude for cathodal tDCS þ rEPNS, cathodal tDCS and sham tDCS þ rEPNS. There was a prominent time effect for cathodal tDCS þ rEPNS (F5,45 ¼ 13.9, P < 0.0001) and cathodal tDCS (F5,45 ¼ 18.1, P < 0.0001) but not for sham tDCS þ rEPNS (P > 0.3). Cathodal tDCS þ rEPNS caused a lasting reduction of MEP amplitudes respect to cathodal tDCS alone (Fig. 2B). Post hoc t tests for cathodal tDCS þ rEPNS revealed a reduction in MEP amplitude at T0 (t1,9 ¼ 8.8, P < 0.0001), T10 (t1,9 ¼ 7.1, P < 0.0001), T20 (t1,9 ¼ 6.2, P < 0.0001), T30 (t1,9 ¼ 4.9, P < 0.0001) and T60 (t1,9 ¼ 4.9,

P < 0.0001) relative to baseline. Instead, post hoc t tests for cathodal tDCS revealed a depression in MEP amplitude at T0 (t1,9 ¼ 6.1, P < 0.0001) alone. Spinal cord excitability Repeated measure ANOVA on H-reflex amplitude revealed a significant interaction between time and conditioning (F8,32 ¼ 5.8, P ¼ 0.0001). Separate follow-up ANOVAs with time as a withinsubject factor showed a prominent time effect for anodal tDCS þ rEPNS (F4,16 ¼ 14.7, P < 0.0001) and sham tDCS þ rEPNS (F4,16 ¼ 8.5, P ¼ 0.0006) but not for anodal tDCS alone (P > 0.6). Anodal tDCS þ rEPNS and sham tDCS þ rEPNS caused a lasting facilitation of H-reflex at T0, T10, T20, T30 (for all measurement P < 0.01) relative to baseline (Fig. 3A). Also, repeated measure ANOVA on H-reflex amplitude revealed a significant interaction between time and conditioning (F8,32 ¼ 3.95, P ¼ 0.002). Separate follow-up ANOVAs with time as a withinsubject factor showed a prominent time effect for cathodal tDCS þ rEPNS (F4,16 ¼ 9.4, P ¼ 0.0004) and sham tDCS þ rEPNS (F4,16 ¼ 8.5, P ¼ 0.0006) but not for cathodal tDCS (P > 0.6). Cathodal tDCS þ rEPNS and sham tDCS þ rEPNS caused a lasting facilitation of H-reflex at T0, T10, T20, T30 (for all measurement P < 0.01) relative to baseline (Fig. 3B). The lasting facilitation of H-reflex for anodal and cathodal tDCS þ rEPNS and sham tDCS þ rEPNS, but not for anodal and

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cathodal tDCS alone, indicated that most likely repetitive electrical stimulation at wrist was capable to modify spinal cord excitability. Moreover, in the five subjects that participated at this control experiment we confirmed the polarity-specific tDCS-induced shift in corticospinal excitability was present also in FCR muscle (see Fig. 4A and B). Short-latency intracortical inhibition (SICI) and intracortical facilitation (ICF)

Short afferent inhibition (SAI) and long afferent inhibition (LAI) Also in this experimental session conditioned MEP amplitudes increased after anodal tDCS þ rEPNS and it decreased after cathodal tDCS þ rEPNS. However, the ratio between unconditioned and conditioned MEP amplitudes did not differ among the blocks of measurements (Table 1). Regarding SAI and LAI rANOVA did not show any main effects of time or conditioning nor an interaction between these factors. Discussion

At baseline, paired-pulse stimulation consistently produced SICI at an ISI of 2 ms and ICF at an interval of 12 ms. The conditioning protocols induced similar changes in amplitude of unconditioned and conditioned MEPs. Conditioned MEP amplitudes increased after anodal tDCS þ rEPNS and it decreased after cathodal tDCS þ rEPNS. However, the ratio between unconditioned and conditioned MEP amplitudes did not differ among the blocks of measurements. For SICI and ICF, there were no main effects of time or conditioning and no interaction between these factors (Table 1). Cortical silent period Neither anodal tDCS þ rEPNS nor cathodal tDCS þ rEPNS induced any significant effect on the duration of the CSP (Table 1). A dedicated rANOVA did not reveal any main effects of time or conditioning nor an interaction between these factors.

We showed that, when tDCS is coupled with a repetitive electrical stimulation of the right median nerve, the result is a longlasting change in the excitability of the homotopic corticospinal output from the stimulated M1. This indexed by a persistent change in MEP amplitudes in the relaxed right APB muscle. The conditioning effects of tDCS þ rEPNS critically depended on the polarity of tDCS because anodal tDCS þ rEPNS produced a marked facilitation of corticospinal excitability whereas cathodal tDCS þ rEPNS caused a depression of corticospinal excitability. A short lasting change (10 min) of the MEP amplitude is produced by the application of 5 min tDCS alone. This is in agreement with previous data from Nitsche and Paulus [10] who demonstrated that, using tDCS intensities similar to those of the present study, a change in MEP amplitude following a 5 min period of anodal or cathodal tDC stimulation persisted for approximately 5e10 min. Repetitive peripheral nerve stimulation alone failed to induce any significant

Figure 4. A) Effects of anodal tDCS þ rEPNS, anodal tDCS and sham tDCS þ rEPNS on MEP amplitude recorded from FCR muscle. Anodal tDCS þ rEPNS induces an increase in MEP amplitude at T0, T10, T20, T30 (for all measurement P < 0.01) relative to baseline. Anodal tDCS alone induces an increase in MEP amplitude only at T0 (P ¼ 0.002). Error bars indicate standard error of the mean. *P  0.05. B) Effects of cathodal tDCS þ rEPNS, cathodal tDCS and sham tDCS þ rEPNS on MEP amplitude recorded from FCR muscle. Cathodal tDCS þ rEPNS induces a decrease in MEP amplitudes at T0, T10, T20, T30 (for all measurement P < 0.01) relative to baseline. Cathodal tDCS induces a depression in MEP amplitude at only at T0 (P ¼ 0.01). Error bars indicate standard error of the mean. *P  0.05.

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Table 1 Other measures of cortical excitability.

RMT (%) AMT (%) ICI (%) ICF (%) SAI (%) LAI (%) CSP (ms)

Conditioning

Baseline

Anodal DC þ rEPNS Cathodal DC þ rEPNS Anodal DC þ rEPNS Cathodal DC þ rEPNS Anodal DC þ rEPNS Cathodal DC þ rEPNS Anodal DC þ rEPNS Cathodal DC þ rEPNS Anodal DC þ rEPNS Cathodal DC þ rEPNS Anodal DC þ rEPNS Cathodal DC þ rEPNS Anodal DC þ rEPNS Cathodal DC þ rEPNS

43 43 37 37.7 64 63 125 127 61 62 53 54 174 178

             

4.8 4 4.5 3.7 17 25 46 31 11 10 13 13 27 24

T0 43 43 37.3 37.8 63 68 133 120 58 64 51 55 172 178

T10              

4.2 3.2 4.4 4 19 19 49 31 17 15 18 11 31 26

42.5 43 36.7 37.4 64 65 127 131 58 63 50 58 173 179

T20              

4.3 4 4.1 4 14 28 28 29 24 20 16 15 30 24

42.8 42.4 36.9 37.2 62 64 122 120 62 68 52 56 174 171

T30              

4.3 4.1 4 4 19 22 20 21 25 17 19 10 25 30

43 42.6 37 37.5 64 62 121 127 59 58 52 58 170 175

T60              

4.1 4.1 4.3 4.1 18 16 16 28 26 24 18 13 24 30

42.5 43 37 37.7 69 69 132 122

       

3.8 4.3 4.2 3.4 19 28 47 24

170  26 170  17

RMT, resting motor threshold; AMT, active motor threshold; SICI, short-latency intracortical inhibition; ICF, intracortical facilitation; CSP, cortical silent period; SAI, shortlatency afferent inhibition; LAI, long-latency afferent inhibition.

changes in MEP amplitude. Indeed previous studies have demonstrated that longer periods of peripheral nerve stimulation (at least 2 h) are needed to induce motor cortical excitability changes [20,21]. However, the lasting facilitation of H-reflex for anodal and cathodal tDCS þ rEPNS and sham tDCS þ rEPNS but not for anodal and cathodal tDCS alone suggests that a even shorter period of repetitive electrical stimulation (5 min) is capable to modify spinal cord excitability. We discuss the relevance of these results to current research that has used transcranial direct current stimulation and peripheral electrical stimulation to induce rapid motor reorganization in the human brain. Mechanism of change Our prediction was that concurrent peripheral stimulation would prolong and potentiate the ‘facilitatory’ anodal tDCS and the ‘inhibitory’ cathodal tDCS after effects. In human subjects tDCS can modify motor cortex excitability dependent on stimulation polarity, intensity and duration [10]. However the mechanism of action of tDCS is not completely understood. In animals, during anodal stimulation cortical neurons are depolarized at a sub-threshold level, while they are hyperpolarized by cathodal tDCS [22]. The respective membrane potential changes could act as a precondition for the induction of after-effects in these animals [23e25]. The fact that the voltage dependent sodium channel blocker carbamazepine (CBZ) eliminates the short-lasting after-effects induced by anodal, but not by cathodal stimulation indicates that similar mechanisms are operating on the human side [26]. In addition, the N-methyl-Daspartate (NMDA)-receptor antagonist dextromethorphan (DMO) suppresses the post-stimulation effects of both anodal and cathodal DC stimulation, strongly suggesting the involvement of NMDA receptors in both types of DC-induced neuroplasticity [26]. Similar to the induction of established types of short- or long-term neuroplasticity, a combination of glutamatergic and membrane mechanisms is necessary to induce the after-effects of tDCS. Liebetanz et al. [26] suggest that polarity-driven alterations of resting membrane potentials represent the crucial mechanisms of the DCinduced after-effects, leading to both an alteration of spontaneous discharge rates and to a change in NMDA-receptor activation. On the other hand, 1500 stimuli of 5Hz-rEPNS at 200% perceptual threshold had no effect on corticospinal excitability. Indeed previous rEPNS studies have already demonstrated that more than 10 min of high-frequency rEPNS are required to provoke consistent increases in corticospinal excitability [9,20,27]. The mechanisms underlying the effects of rEPNS on motor cortical function are still under investigation, but may include LTP-like phenomena at the

level of cortical synapses [20,28] or a modulation of GABAergic interneurons activity [29]. It is worthy to note that rEPNS (30e180 min) applied to one body part can also modulate BOLD activity in its motor cortical representation in M1 and possibly in the dorsal premotor cortices [30,31]. The mechanisms underlying the long lasting effects of this protocol employing tDCS paired with peripheral electrical stimulation are currently unknown. PAS occurs when an input to a post-synaptic cell is synchronous a postsynaptic depolarization provided by intracellular injected depolarizing currents in the post-synaptic cell [5]. These observations in animal models suggest that associative LTP induction requires two events: synapse activation and depolarization of the post-synaptic membrane [32,33]. A possible explanation of our results is that our neurophysiological protocol uses a principle similar to that leading to associative LTP in previous studies on animal cortical slices by delivering an input to a post-synaptic cell synchronous to a post-synaptic depolarization. Based on this model, if median nerve rEPNS reaches the somatosensory and motor cortex at a time when the membrane potential of its pyramidal neuron is modified by tDCS, this would produce more effective changes of motor cortex excitability of those induced by tDCS alone. In a previous paper, Uy and co-workers (2003) showed that a short period (5 min) of weak anodal direct current stimulation prior to a period (10 min) of peripheral ulnar nerve stimulation was capable to induce a significant and persistent increase in motor cortical excitability [34]. The authors suggest that the changes induced in the study where anodal DC stimulation and peripheral nerve stimulation are applied successively are most likely to be due to an LTP like mechanism [34]. In the present electrophysiological approach, at a difference with the Uy’s conditioning protocol, the period of tDCS was simultaneous (5 min) with the repetitive electrical stimulation of peripheral nerve. As an alternative explanation we can hypothesize that afferent sensory inputs activates multiple set of inhibitory intra-cortical circuits (for instance those producing SAI and LAI). Concurrent tDCS might have a polarity-specific effect on how efficiently 5Hz-rEPNS can activate these inhibitory intra-cortical circuits. This in turn might have a gating effect on the polarityspecific induction of LTP or LTD like effects during tDCS. However this hypothesis is unlikely since both anodal tDCS þ rEPNS and cathodal tDCS þ rEPNS did not interact with SICI, ICF, CSP, SAI and LAI. Site of change Several evidences suggest that tDCS directly affects the underlying cortex and that cortical neurons alter their activity state

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during, as well as after, the current flow [22,23,35e37]. Further indication for the site of action of tDCS originates from the paper of Liebetanz and co-workers [26]. They studied a sequence of transcranial electrical stimulation (TES) and TMS-elicited MEPs before and after tDCS. In contrast to TMS, TES produces mainly a direct activation of pyramidal neurons at sub-cortical level [38,39]. These authors showed that the TES-evoked MEPs did not vary after 5 min of tDCS. The results of this study point out that the TMS-assessed changes in motor cortical excitability are most probably localized within gray matter of the motor cortex and not at sub-cortical level [26]. Moreover Nietsche and co-workers demonstrated that Hreflex and F-waves, neurophysiological measurements that reflect the excitability of the second motor neuron, did not change during tDCS [11]. These data confirm that tDCS after-effects are localized proximal to the spinal motor neuron. In contrast, recently a paper of Roche and associates investigated reciprocal inhibition after 20 min of tDCS [40]; these authors showed that anodal but not cathodal tDCS increases disynaptic inhibition directed from extensor carpi radialis (ECR) to flexor carpi radialis (FCR) with no modification of presynaptic inhibition of FCR Ia terminals and FCR H-reflex recruitment curves. Hence they concluded that tDCS may act either on cortical and spinal circuits [40]. The lasting facilitation of H-reflex observed for anodal and cathodal tDCS þ rEPNS and sham tDCS þ rEPNS indicates that 5 Hz peripheral electrical stimulation could modify spinal cord excitability. However it cannot be excluded that 5Hz-rEPNS could act on a motor neuronal pool which is different from the one activated by TMS. This could explain the H reflex modifications without changed in MEP amplitude after peripheral stimulation alone. Nevertheless, to the best of our knowledge we are not aware of any report in literature that evaluated the modification of H-reflex after longer periods of peripheral nerve stimulation. Therefore we can conclude that the after effects of this protocol could be related to a mixture of cortical and spinal mechanisms. Future implications Functional recovery from stroke is variable and the mechanisms underlying recovery are not well understood. In recent years, different forms of non-invasive brain stimulation techniques have been employed in order to boost up plasticity and recovery. Among them tDCS has generated excitement as a potential neurorehabilitative adjuvant strategy to improve motor performances and language skills in stroke patients [41e43]. Moreover, peripheral nerve stimulation has also been proposed as an additional strategy capable of ameliorating pinch strength [44], swallowing [45], and ADL-like tasks [46] in stroke patients [47]. A recent paper of Celnik and co-workers showed that combining peripheral nerve stimulation (2 h) delivered on the paretic hand with anodal tDCS (20 min) over the ipsilesional M1, in association with motor training, induces an additional improvement in motor performance compared to the use of each stimulation type alone in combination with sham and training [48]. These findings would suggest that combining peripheral nerve stimulation with tDCS along with physical practice could represent a future better strategy in neurorehabilitation. References [1] Bliss TV, Lomo T. Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. J Physiol 1973;232:331e56. [2] Huang YZ, Edwards MJ, Rounis E, Bhatia KP, Rothwell JC. Theta burst stimulation of the human motor cortex. Neuron 2005;45:201e6. [3] Markram H, Lubke J, Frotscher M, Sakmann B. Regulation of synaptic efficacy by coincidence of postsynaptic APs and EPSPs. Science 1997;275:213e5.

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Increased transcranial direct current stimulation after effects during concurrent peripheral electrical nerve stimulation.

In this study we tested the hypothesis whether a lasting change in the excitability of cortical output circuits can be obtained in healthy humans by c...
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