Boosting brain excitability by transcranial high frequency stimulation in the ripple range

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1 J Physiol (2010) pp Boosting brain excitability by transcranial high frequency stimulation in the ripple range Vera Moliadze, Andrea Antal and Walter Paulus Department of Clinical Neurophysiology, Georg-August University, Robert-Koch-Straße 40, 37075, Göttingen Alleviating the symptoms of neurological diseases by increasing cortical excitability through transcranial stimulation is an ongoing scientific challenge. Here, we tackle this issue by interfering with high frequency oscillations ( Hz) via external application of transcranial alternating current stimulation (tacs) over the human motor cortex (M1). Twenty-one subjects participated in three different experimental studies and they received on separate days tacs at three frequencies (80 Hz, 140 Hz and 250 Hz) and in a randomized order. tacs with 140 Hz frequency increased M1 excitability as measured by transcranial magnetic stimulation-generated motor evoked potentials (MEPs) during and for up to 1 h after stimulation. Control experiments with sham and 80 Hz stimulation were without any effect, and 250 Hz stimulation was less efficient with a delayed excitability induction and reduced duration. After-effects elicited by 140 Hz stimulation were robust against inversion of test MEP amplitudes seen normally under activation. Stimulation at 140 Hz reduced short interval intracortical inhibition, but left intracortical facilitation, long interval cortical inhibition and cortical silent period unchanged. Implicit motor learning was not facilitated by 140 Hz stimulation. High frequency stimulation in the ripple range is a new promising non-invasive brain stimulation protocol to increase human cortical excitability during and after the end of stimulation. (Resubmitted 27 July 2010; accepted after revision 16 October 2010; first published online 20 October 2010) Corresponding author V. Moliadze: Department of Clinical Neurophysiology, Georg-August University, Robert-Koch-Straße 40, Göttingen, Germany. vmoliadze@med.uni-goettingen.de Abbreviations AMT, active motor thresholds; CSP, cortical silent period; EMG, electromyogram; FDI, first dorsal interosseous muscle; ICF, intracortical facilitation; itbs, intermittent theta burst stimulation; M1, primary motor cortex; MEP, motor evoked potential; PAS, paired associative stimulation; PPTMS, paired-pulse transcranial magnetic stimulation; RMT, resting motor threshold; SICI, short intracortical inhibition; SRTT, serial reaction time task; tacs, transcranial alternating current stimulation; tdcs, transcranial direct current stimulation; TMS, transcranial magnetic stimulation; trns, transcranial random noise stimulation. Introduction Specific linkages have been drawn between neuronal oscillations in defined frequency bands and a variety of cognitive functions and sensory selection (for a recent review see: Schroeder & Lakatos, 2009). One subgroup of high frequency oscillations are known as ripples, a term that generally refers to oscillations occurring at about Hz. This subgroup has been well described in the hippocampus and parahippocampal structures and plays a key role both in normal behaviours and abnormal brain functionssuchasepilepsy (Buzsakiet al. 1992; Ylinen et al. 1995; Grenier et al. 2001, 2003; Diba & Buzsaki, 2007; Middleton et al. 2008; Engelet al. 2009). Another subset, named fast ripples, comprises oscillations from 250 to 500 Hz, and has been proposed as a bio-marker of epileptic tissue (Bragin et al. 1999; Jacobs et al. 2009). These fast ripples have been recorded also in healthy neocortex but not in normal hippocampus and parahippocampal structures (Engel et al. 2009). Some cortical neurons are able to generate even higher frequencies of up to 800 Hz (Steriade et al. 1993; Amassian & Stewart, 2003). So far interest in human oscillatory non-invasive brain stimulation has mainly focused on the traditional EEG frequency bands (Kanai et al. 2008; Pogosyan et al. 2009) and on higher gamma oscillations targeting aspects of cortical binding (Singer, 2009). Expanding transcranial stimulation frequencies in the range above 100 Hz is expected to be of importance to engender research in several directions. It represents a further step towards more targeted plasticity modulating protocols and also may have important implications for neurological disease accompanied by pathological oscillations and motor DOI: /jphysiol

2 4892 V. Moliadze and others J Physiol deficits. Interestingly frequencies in the ripple range are intensively used in deep brain stimulation for treatment of Parkinson s disease. Direct frequency-dependent effects of Hz on afferents to the subthalamic nucleus region have become a major direct target of deep brain stimulation in PD (Hammond et al. 2007; Gradinaru et al. 2009). Here we modulate motor cortex excitability by transcranial high frequency alternating current stimulation (tacs) with a frequency of 140 Hz whichisclosetothisfrequencyandisinthemiddleofthe low ripple frequency range. As control conditions we explored the effects of 80 and 250 Hz oscillations on motor cortex excitability. These frequencies represent the lower and higher border of the ripple range, and therefore we hypothesized that tacs stimulation with these frequencies would be less effective for changing cortical excitability than applying 140 Hz stimulation. Furthermore, in a recent study it was described that frequencies below 100 Hz did not modify intracortical and corticospinal excitability in a random noise stimulation protocol (Terney et al. 2008). For evaluating cortical excitability changes we applied several transcranial magnetic stimulation (TMS) protocols that are known to test the functions and integrity of specific cortical circuits in the human motor network. The change in amplitude of the motor evoked potential (MEP) elicited by single pulse TMS represents the global change of corticospinal excitability in the motor system. As global parameters of cortico-spinal excitability we measured the input output (I-O) curve and motor thresholds (Chen, 2000; Abbruzzese & Trompetto, 2002). Short intracortical inhibition (SICI) and facilitation (ICF) were studied using a paired-pulse stimulation protocol (Kujirai et al. 1993). Here a subthreshold TMS stimulus (conditioning pulse) was followed by a suprathreshold test stimulus. The resulting increase or decrease of the MEP amplitude elicited by the test stimulus is determined by the interstimulus interval (ISI): short ISIs (2 and 4 ms) and long ISIs (7, 9 and 12 ms). Furthermore we measured the long interval cortical inhibition (LICI) with two suprathreshold stimuli applied with 50, 100 and 150 ms stimulation intervals, (Valls-Sole et al. 1992). We also measured cortical silent period (CSP), which is commonly used to measure cortical and spinal inhibitory functions (Fuhr et al. 1991; Paulus et al. 2008). In addition, we studied plastic after-effect changes under muscular activation in comparison to the effects in rest. Both abolition (Huang et al. 2008b) and reversal (Antal et al. 2007; Terney et al. 2008) of motor cortical excitability changes, induced by either intermittent theta burst stimulation (itbs) (Huang et al. 2008b), transcranial direct current stimulation (tdcs) (Antal et al. 2007) or transcranial random noise stimulation (trns) (Terney et al. 2008), have been describedunderactivation. Here we show that ripple stimulation after-effects are comparatively robust after switching the innervation state of the target muscle from rest to activation. Furthermore, a behavioural task was used to study ripple oscillation-driven changes in performance during a variant of the serial reaction time task (SRTT) (Nissen & Bullemer, 1987), which is a standard protocol to test implicit motor learning. We hypothesized that externally applied high frequency oscillation in the ripple range will interfere with ongoing oscillations and neuronal activity in the brain and therefore result in significant changes of cortical and corticospinal excitability. Methods The study conformed to the Declaration of Helsinki, and the experimental protocol was approved by the Ethics Committee of the University of Göttingen. Subjects In total 21 subjects (age 25.9 ± 2.35 S.D. years, range: years) participated in this study (for details see Table 1). All subjects were right handed, according to the Edinburgh handedness inventory (Oldfield, 1971), and they were naive with regard to the aim of the study. Those who were ill, pregnant, suffering from drug abuse, or had metallic implants/implanted electrical devices were excluded by an interview and a short physical examination. All gave written informed consent. Subjects, but not the investigator, were blinded for stimulation conditions in all of the studies. Electrical stimulation of the motor cortex Electrical stimulation was delivered by a battery-driven stimulator (NeuroConn GmbH, Ilmenau, Germany). tacs was applied for 10 min with a current intensity of 1000μA. The waveform of the stimulation was sinusoidal without DC offset. The current was ramped up and down over the first and last 5 s of stimulation. For sham stimulation, the current was turned on for 5 s at the beginning and 5 s at the end of the stimulation. Since high frequency oscillations in the ripple range did not induce a flickering sensation, subjects were kept blinded with regard to the type of the experiment. The stimulating electrodes were attached to the scalp using electrode Ten20 EEG conductive paste. They were further fixed with a rubber band placed over the electrode and attached under the chin. This reduced electrode impedance and prevented movement of the electrodes during the experiment. Sufficient cream ensured low impedance during the stimulation time. The impedance was kept at <5k. The reference electrode was placed

3 J Physiol High frequency oscillations and brain excitability 4893 Table 1. Stimulations protocols, electrode sizes, subjects characteristics and baseline values for the performed experiments Baseline (single TMS) and test pulse (pptms) Electrode AC stimulated Number of amplitudes (mv) ± Experiment Measurement size muscle subjects S.E.M. Sex: (f/m) Experiment 1 Single pulse S: 16 cm 2 ; R: Right FDI Sham: ± /6 tac stimulation TMS 84 cm 2 80 Hz: ± /5 under complete 140 Hz: 12 1 ± /6 muscle relaxation 250 Hz: 8 6 ± /5 I-O, SICI/ICF, S: 16 cm 2 ; R: Right FDI Sham: 9 3 ± 0.04 LICI, CSP 84 cm 2 80 Hz: ± /4 140 Hz: ± Hz: 9 0 ± 0.03 Experiment 2 Single pulse S: 16 cm 2 ; R: Right FDI Sham: 9 1 ± 0.02 tac stimulation TMS 84 m 2 80 Hz: 9 0 ± /3 under motor 140 Hz: 9 3 ± 0.03 activation 250 Hz: 9 0 ± 0.03 Experiment 3 S: 16 cm 2 ;R: Sham:13 Behavioural studies 84 cm 2 80 Hz: 13 5/8 (SRTT) 140 Hz: Hz: 13 Baseline MEP amplitude means of about 1 mv were calculated for each experimental condition. The single test-pulse TMS intensity was adjusted to achieve a baseline MEP of SI 1mV, and re-adjusted during the respective stimulation protocol if needed, to compensate for effects of global excitability changes on test-pulse amplitude. They did not differ between the respective conditions (Student s t test, P < 0.05). f, female; m, male; R, reference electrode (frontopolar); S, motor cortex stimulation electrode; FDI, first dorsal interosseous muscle. in a saline soaked sponge. To increase the focality of the stimulation, the electrode size was 4 4cmwhereasthe reference electrode size at the forehead was 14 6cm.In each experiment, the motor-cortical electrode was fixed over the representational field of right first dorsal interosseous (FDI), as identified by TMS. Measurement of motor system excitability To detect stimulation-driven changes of excitability, MEPs of the motor cortical representation of the right FDI were recorded during and following stimulation. The site of the stimulation was determined using single pulse TMS. MEPs were elicited using a Magstim 200 magnetic stimulator (Magstim Company, Whiteland, Wales, UK) with a figure-of-eight standard double magnetic coil (diameter of one winding, 70 mm; peak magnetic field, 2.2 T; average inductance, μh). The coil was held tangentially to the skull, with the handle pointing backwards and laterally at 45 deg from the midline, resulting in a posterior anterior direction of current flow in the brain. The coil was connected to two monophasic Magstim 200 stimulators via a bistim module during the paired-pulse TMS study. Surface EMG was recorded from the right FDI through a pair of Ag AgCl surface electrodes in a belly-tendon montage. Raw signals were amplified, band-pass filtered (2 Hz to 2 khz; sampling rate, 5 khz), digitized with a micro 1401 AD converter (Cambridge Electronic Design, Cambridge, UK) controlled by Signal Software (Cambridge Electronic Design, version 2.13), and stored on a personal computer for off-line analysis. The FDI hotspot was defined as the site where TMS resulted consistently in the largest MEP in the resting muscle. For single pulse TMS studies in experiment 1, the TMS coilwasplacedovertheelectrode/rubbersupportband montage and TMS stimulation intensity was adjusted to achieve a baseline MEP of about 1 mv. Experimental design Subjects participated in three different experimental studies and they received on separate days tacs (80 Hz, 140 Hz and 250 Hz) and s. The order of the stimulations with regard to all experiments occurred in a counterbalanced fashion. Experiment 1. tac stimulation under relaxed condition Single pulse TMS study. Twelve healthy volunteers participated in this study. All of them took part in the sham and 140 Hz stimulation experiments, while eight

4 4894 V. Moliadze and others J Physiol subjects participated in studies performed with 80 and 250 Hz. The experiments were performed with at least 5 days in between and at the same time of the day for each subject. tac stimulation was performed under complete relaxation, which was controlled through visual feedback of electromyogram (EMG) activity. During stimulation we have recorded TMS elicited MEPs. Twenty single test-pulse MEPs were recorded at 0, 5 and 10 min after stimulation andthenevery10minupto60min. The study with regard to I-O curve, SICI/ICF, LICI, CSP protocols. To explore the origin of the excitability modulation in more detail, we measured the input output curve and motor thresholds as global parameters of cortico-spinal excitability, and determined short intracortical inhibition and intracortical facilitation as well as long interval cortical inhibition and cortical silent period. Nine subjects participated in four experimental sessions on separate days. Stimulus intensities (as a percentage of maximal stimulator output) of TMS were determined at the beginning of each experiment. SI 1mV (the intensity required to evoke MEPs of 1 mv peak-to-peak amplitude) was determined with single-pulse TMS first (the amplitude of the test MEP was matched before and after tacs). Motor threshold determination The rest motor threshold (RMT) was determined as the minimum stimulator output needed to produce a response of at least 50 μv in the relaxed FDI in at least 3 of 6 consecutive trials. The active motor threshold (AMT) was defined as the lowest stimulus intensity at which 5 out of 10 consecutive stimuli elicited reliable MEPs (above 200μV in amplitude) during isometric contraction of the contralateral FDI muscle (Rothwell et al. 1999) in at least 3 of 6 recordings. Input output curve I-O curve were measured with three different and increasing stimulus intensities (110%, 130% and 150% of RMT), each with 10 pulses. A mean was calculated for all intensities. Short intracortical inhibition and facilitation For SICI/ICF, two magnetic stimuli were given through the same stimulating coil, and the effect of the first (conditioning) stimulus on the second (test) stimulus was investigated (Kujirai et al. 1993). To avoid any floor or ceiling effect, the intensity of the conditioning stimulus was set to a relatively low value of 80% of AMT. The single test-pulse TMS intensity was adjusted to achieve a baseline MEP of SI 1mV, and re-adjusted during the respective stimulation protocols, to compensate for effects of global excitability changes on test-pulse amplitude caused by tacs. SICI was taken as the mean percentage of inhibition at ISIs of 2 and 4 ms, whereas ICF was taken as the mean facilitation at ISIs of 7, 9 and 12 ms. The control condition (test pulse alone) was tested 20 times, and each of the conditioning-test stimuli 10 times. The mean peak-to-peak amplitude of the conditioned MEP at each ISI was expressed as a percentage of the mean peak-to-peak size of the unconditioned test pulse. Long intracortical inhibition We tested LICI with two suprathreshold stimuli applied with ISIs of 50, 100, 150 and 200 ms (Valls-Sole et al. 1992). The intensity of both stimuli was set to 110% of RMT. Here as well, the intensity was set to this relatively low value to avoid any floor or ceiling effect. The control condition (first pulse alone) was tested 20 times, whereas each of the paired stimuli was tested 10 times. LICI was taken as the mean percentage inhibition of conditioned MEP at ISIs of 50, 100, 150 and 200 ms. Cortical silent period Ten pulses with SI 1mV and 10 pulses with 120% RMT were applied under tonic contraction of the right FDI muscle. The subjects had to squeeze a ball with a size of 8 cm connected to a display on which the actual pressure values of the ball were quantified. They were instructed to maintain a voluntary contraction of about 20% of maximum voluntary force using visual feedback. CSPs were separately determined; in rectified and averaged EMG traces with a prestimulus period of 100 ms. CSP (in ms) was measured from the TMS stimulus to the point where the signal reached the amplitude of the mean prestimulus EMG activity. Experiment 2. Motor task-related modulation of different frequency tacs Single pulse TMS study. In this experiment nine subjects were instructed to squeeze a ball (8 cm diameter) in their right hand during tacs. The ball was connected to a display where the actual values related to pressure were quantified. Before the stimulation session, the subjects were asked to push the ball as hard as possible. During the tacs session, subjects had to squeeze the ball to half-maximal contraction as previously shown. Due to EMG contamination during squeezing we confined our measurements of MEP responses to the relaxed state after tacs; 20 single test-pulse MEPs were recorded at 0, 5

5 J Physiol High frequency oscillations and brain excitability 4895 and 10 min after stimulation and then every 10 min up to 60 min. Experiment 3. Behavioural studies (SRTT) Thirteen subjects participated in the behavioural experiment. All subjects received sham, 80 Hz, 140 Hz and 250 Hz stimulation in different sessions separated by at least 1 week to prevent carryover effects of task learning and stimulation. The order of the different stimulation conditions was randomized between subjects and they received different sequences for SRTT. Subjects were seated in front of a computer screen at eye level behind a response pad with four buttons numbered 1 4 and were instructed to push each button with a different finger of the right hand (index finger for button 1, middle finger for button 2, ring finger for button 3, and little finger for button 4). An asterisk appeared in one of four positions that were horizontally spaced on a computer screen and permanently marked by dots. The subjects were instructed to press the key corresponding to the position of the asterisk as fast as possible. After a button was pushed, the go signal disappeared. The next go signal was displayed 500 ms later. The test consisted of eight blocks of 120 trials. In blocks 1 and 6, the sequence of asterisks followed a pseudorandom order in that asterisks were presented equally frequently in each position and never in the same position in two subsequent trials. In blocks 2 5, 7 and 8, the same 12-trial sequence of asterisk positions was repeated 10 times. Subjects were not informed about the repeating sequence. Analysis and statistics In the first stage of analysis, the power analysis was conducted for each experiment separately using the Statistica software 7.1 (StatSoft Inc., Tulsa, OK, USA). According to the mean difference in MEP amplitudes between the sham and tacs treated groups based on preliminary studies, n > 7 at each group is sufficient to detect the relevant difference with 80% power at an α level of 0.05 (two-sided) using repeated measures ANOVA. Single pulse TMS studies. First the TMS intensity resulting in MEP amplitudes of 1 mv was established. The baseline of TMS-evoked MEPs (20 stimuli) was recorded at 0.25 Hz before the stimulation. 20 single test-pulse MEPs were recorded at 0, 5 and 10 min after stimulation and then every 10 min up to 60 min. In experiment 1 (Single pulse TMS studies) the MEPs measured during tacs were analysed in successive groups of 15, each covering a time range of 1 min, and the means for each group were calculated. In the first experiment a repeated measures ANOVA with the within subject factor time, the inbetween subject factor tacs, and the dependent variable MEP amplitude has been used (TYPE OF STIMULATION 4levels TIME 19 levels, independent variables time course before, during and after electrical stimulation and frequency of stimulation; dependent variable MEP amplitude). In Experiment 2 a repeated measures ANOVA was performed with TYPE OF STIMULATION 4levels TIME 9levels (independent variables: time course before and after electrical stimulation and frequency of stimulation; dependent variable: MEP amplitude). In the case of a significant main effect of TYPE OF STIMULATION or the interaction of TIME and TYPE OF STIMULATION, Fisher s least significant difference (LSD) test was performed. The study with regard to I-O curve and SICI/ICF, LICI and CSP protocols. First intra-individual MEP amplitudes means were calculated for the TMS stimulation conditions, TMS intensity with regard to I-O curve and CSP and ISI with regard to SICI/ICF and LICI. For paired-pulse stimulation protocols, the resulting means were standardized to the respective single-pulse condition. Then inter-individual means were calculated for each condition. In order to determine significant changes related to a given stimulation condition, repeated-measures ANOVAs were performed, entering four level ANOVA; independent variables: ISI/stimulation intensities and type of stimulation, dependent variable: MEP amplitude. In the case of a significant type of stimulation or interaction between ISI/intensity and type of stimulation, Fisher s LSD test was performed. Implicit learning. Concerning the implicit learning (Experiment 3) statistical analysis was performed using repeated measures ANOVA (type of stimulation 8 blocks) for reaction time (RT), error rate (ER), and variability. Because the RT and ER differences between Blocks 5 and 6 are thought to represent an exclusive measure of implicit learning, an interactive Student s t test was performed to compare the respective differences for the tac stimulation condition versus sham condition. An ER was calculated to assess the number of incorrect responses for each block and each subject in each stimulation condition. Safety aspects All subjects completed a questionnaire, which contained rating scales regarding the presence and severity of headache, difficulties in concentrating, acute mood changes, and any discomforting sensation like pain,

6 4896 V. Moliadze and others J Physiol tingling, itching or burning under the electrodes, during and after stimulation. None of them reported any side effects. In a previous study, Terney et al. (2008) showed that the concentration of the serum neuron-specific enolase (NSE), a sensitive marker of neuronal damage, was unchanged after trns. In the present study we used frequencies from 80 to 250 Hz, which are in the range of Hz. The study with regard to I-O curve and SICI/ICF, LICI and CSP protocols. Input output curve. As shown in Fig. 2, 140 Hz tacs increased the slope of the I O curve. Controls by sham, 80 and 250 Hz stimulation were without any effect. The repeated measures of ANOVA revealed a significant main effects of TYPE OF STIMULATION (F 3.24 = 3.73, P = 0.02) and INTENSITY (F 2.16 = 39.10, P < 0.01). The interaction between TYPE OF STIMULATION and INTENSITY was not significant (F 6.48 = 1.11, P = 0.4). Results Experiment 1. tac stimulation under complete muscle relaxation Single pulse TMS study. The repeated measures of ANOVA revealed a significant main effects of TYPE OF STIMULATION (F 3.36 = 14.64, P < ) and TIME (F = 5.07, P < ). The interaction between TYPE OF STIMULATION and TIME was also significant (F = 2.41, P < ). The most important finding of this experiment was that when 140 Hz tacs was applied to the M1, cortical excitability increased up to 44% above baseline. According to Fisher s LSD test, significantly increased MEPs were observed with 140 Hz tacs between 2 min and 10 min during stimulation (ST2-ST10) and post-stimulation up to 60 min (PST0 PST60) compared to (P < 0.005). We compare MEP amplitudes at the single time points during and post-stimulation with baseline MEP amplitudes. tacs of 140 Hz applied with 1 ma intensity induced a significant elevation in MEP amplitude compared to baseline at the time points ST2 ST10 and PST0 PST60 (Fisher s LSD, P < 0.005). See Fig. 1A. In contrast to the effect of 140 Hz stimulation, 80 Hz AC stimulation did not modify the MEP amplitudes significantly, when compared with. See Fig. 1B. According to Fisher s LSD analysis, 250 Hz AC stimulation induced a significant increase of MEPs compared to the at time points ST6 ST10 (P < 0.005) and PST0 PST5 (P < 0.005).This short-duration facilitation was followed by a decline in excitation back to baseline. Compared to baseline, MEPs were increased at the time points PST6 PST10 (P = 0.02) and PST0 PST5 (Fisher s LSD, P < 0.05). See Fig. 1C. Post hoc tests showed that the 140 Hz tacs applied with 1 ma intensity induced a significant elevation in MEP compared to 80 Hz and 250 Hz stimulation (Fisher s LSD, P < 0.05), while 250 Hz did not modify MEP amplitudes significantly when we compared with 80 Hz stimulation. See Fig. 1D. Short intracortical inhibition and facilitation. The ANOVA revealed a significant effect of TYPE OF STIMULATION (F 3.24 = 3.15, P = 0.04). However, the interaction between TYPE OF STIMULATION and ISI was not significant (F 3.24 = 0.63, P = 0.6). According to the post hoc analysis, MEPs were significantly less decreased at ISI of 2 and 4 ms after 140 Hz stimulation compared with the sham condition. P < 0.05 (See Fig. 3). Administration of 140 Hz had no effect on ICF, LICI and CSP as revealed by the results of the respective ANOVAs (Table 2). Experiment 2. Motor task-related modulation of different frequency tacs Single pulse TMS study. In previous publications, muscle contraction decreased MEP observed after mental effort and motor activation using tdcs (Antal et al. 2007), paired associative stimulation (PAS) (Stefan et al. 2004), theta burst stimulation (TBS) (Huang et al. 2008a) and trns (Terney et al. 2008). In this study for the first time combined with muscle activation was recorded. Muscle contraction itself results in a significant decrease in MEP amplitude compared to baseline observed at the time points PST0 PST10 (Fisher LSD, P < 0.01). Baseline comparisons for each verum stimulation showed suppression of MEP amplitudes at the 0 time point (P < 0.05). See Fig. 4. tacs of 80 Hz and 250 Hz did not modify the activation-induced decrease of MEP size; only 140 Hz resulted in a MEP size increase. Repeated measures ANOVA revealed a significant effect of TYPE OF STIMULATION (F 3.24 = 4.87, P = 0.009) and TIME (F 8.64 = 7.06, P < ). The interaction between TYPE OF STIMULATION and TIME was not significant (F = 1. 29, P = 0.2). According to Fisher s LSD analysis, significantly increased MEPs were observed with 140 Hz at the PST5 PST50 time points compared to the time point using (P < 0.05). See Fig. 4.

7 J Physiol High frequency oscillations and brain excitability 4897 Experiment 3. Behavioural studies (SRTT) No improvement of reaction times was evident during 140Hz,incontrasttotDCS(Nitscheet al. 2003) and to trns (Terney et al. 2008). For reaction time the repeated measures ANOVA revealed a significant main effect of blocks (F 7.77 = 20.43, P < ); however the main effect of TYPE OF STIMULATION (F 3.33 = 0.83, P = ) and interaction between TYPE OF STIMULATION and BLOCKS (F = 1.43, P = 0.1) were not significant. Interactive t tests revealed no significant differences between blocks 5 and 6 between active and sham conditions. 2.0 A 80 Hz stimulation 2.0 B 140 Hz stimulation During Stimulation After Stimulation During Stimulation After Stimulation before ST1 ST2 ST3 ST4 ST5 ST6 ST7 ST8 ST9 ST10 PST0 PST5 PST10 PST20 PST30 PST40 PST50 PST60 -- before ST1 ST2 ST3 ST4 ST5 ST6 ST7 ST8 ST9 ST10 PST0 PST5 PST10 PST20 PST30 PST40 PST50 PST C 250Hz stimulation 2.0 D During Stimulation After Stimulation before ST1 ST2 ST3 ST4 ST5 ST6 ST7 ST8 ST9 ST10 PST0 PST5 PST10 PST20 PST30 PST40 PST50 PST60 80 Hz Stimulation 140 Hz Stimulation 250 Hz Stimulation Figure 1. tac stimulation under complete muscle relaxation A, 140 Hz AC stimulation significantly increased MEPs at the ST2 ST10 and PST0 PST60 time points compared to the ( P < 0.05). B, controls by sham and 80 Hz stimulation were without any effect. C, 250 Hz AC stimulation significantly increased MEPs at the ST6 ST10 and PST0 PST5. The figure shows mean amplitudes of MEPs and their S.E.M. during 10 min and after tac stimulation up to 60 min. Filled symbols indicate significant deviations of the during- and post-measurements of MEP amplitudes from baseline values; P < D, recalculated data of A, B and C in order to sum up the 1 ma tacs induced effects on cortical excitability. Different frequencies show different behaviours. Using 140 Hz stimulation MEP amplitudes increased most. Post hoc tests showed that the 140 Hz tacs applied with 1 ma intensity induced a significant elevation in MEP compared to 80 Hz and 250 Hz stimulation (Fisher s LSD test, P < 0.05). Error bars indicate S.E.M. The bar graphs show the MEP value from ST1 to PST60. P < 0.05.

8 4898 V. Moliadze and others J Physiol As shown in Fig. 5C, with 250 Hz a slight improvement was evident in Blocks 2 5. Figure 5 shows the differences between 80 Hz, 140 Hz and 250 Hz tacs and. For the error rate (ER) in all conditions, the ANOVAs showed a significant main effect on blocks. Despite this, the results of all other tests in all condition remained non-significant. Discussion Here we demonstrate for the first time that high frequency oscillatory non-invasive brain stimulation is capable of inducing frequency-specific long-lasting cerebral excitability elevations in humans. Using 10 min tacs at the peak ripple frequency of 140 Hz we could increase M1 excitability as measured by TMS immediately and for at least an hour afterwards. Controls of sham and 80 Hz stimulation were without any effect. A 10 min application of 250 Hz also elicited a less prominent increase of MEP amplitudes, which predominates later during the stimulation and persisted for a shorter time post-stimulation compared to 140 Hz tacs. Support for the assumption that tacs in a frequency range above 100 Hz is effective comes from a recent study in the dorsal column pathway using epidural stimulation as a tool to restore locomotive capability in animal models of Parkinson s disease symptoms. In this study the effect was strongest for 300 Hz stimulation (Fuentes et al. 2009). Taking into account our previous studies on tdcs, tacs at 140 Hz stimulation duration seems to be at least as effective as anodal tdcs (Nitsche & Paulus, 2001) or trns (Terney et al. 2008) applied at the same intensity and stimulation duration of 1 ma and 10 min. However, tacs avoids the direction sensitivity that is observed by using tdcs and goes along completely unnoticed by the subjects. In contrast to tdcs so far we see no possibility of reducing corticospinal excitability with 140 Hz tacs of the motor cortex. tacs of 140 Hz has a better blinding potential for controlled studies compared to rtms or tdcs. It confirms also the sensitivity to high frequency stimulation seen in human cortical excitability studies with trns (Terney et al. 2008). In this study a random noise frequency range between 100 and 640 Hz was responsible for excitatory after-effects, in contrast to the frequency range below 100 Hz, which was also ineffective, such as the 80 Hz condition here. It remains to be determined if the trns result was essentially based on inducing a resonance 3 80 Hz stimulation Hz stimulation % 130% 150% RMT 0 110% 130% 150% RMT Hz stimulation % 130% 150% RMT Figure 2. tacs of 140 Hz under complete muscle relaxation shifts the slope of the input output curve The MEP amplitudes (means ± S.E.M.) at 110, 130 and 150% of resting MT (RMT) are shown for sham, 80, 140 and 250 Hz tac stimulations. P < 0.05, Fisher s LSD test.

9 J Physiol High frequency oscillations and brain excitability 4899 phenomenon in the ripple frequency range. Not only DC fields, but also AC fields may cause polarization of cell membranes (Deans et al. 2007). Since the membrane is loaded with lots of voltage gated channels, which are non-linear, these induced changes in membrane fluctuation are not just cancelled out, but may even be amplified and result in a net depolarization. Further differences from transcranial magnetic or direct current stimulation results can also be observed. Excitability increase under motor activation was reduced with 140 Hz tacs but not inverted such as seen in both anodal and cathodal tdcs (Antal et al. 2007). In contrast to anodal tdcs implicit motor learning was not significantly facilitated with tacs at any of the three frequencies used here. Obviously 140 Hz tacs is less effective for facilitating implicit learning than both trns (Terney et al. 2008) or anodal tdcs (Nitsche et al. 2003) since in the present study there was only a tendency for a improvement in reaction times in the late blocks 7 and 8, for 250 Hz. The non-significant changes concerning implicit learning were probably caused by a too low number of subjects; larger sample sizes would have been necessary to prove the significant effect of stimulation if there is any. In our previous study applying high frequency trns, we have found an increased ICF after trns over M1 using the paired-pulse protocol (Terney et al. 2008). 2.5 A 80 Hz stimulation 2.5 B 140 Hz stimulation Double pulses/tets pulse MEP amplitude 2.0 Double pulses/tets pulse MEP amplitude ISI 2 ISI 4 ISI 7 ISI 9 ISI 0.0 ISI 12 ISI 2 ISI 4 ISI 7 ISI 9 ISI 12 ISI 2.5 C 250Hz stimulation 2.5 D Double pulses/tets pulse MEP amplitude 2.0 Double pulses/tets pulse MEP amplitude ISI 2 ISI 4 ISI 7 ISI 9 ISI 12 ISI 0.0 SICI ICF 80 Hz stimulation 140 Hz stimulation 250 Hz stimulation Figure 3. Intracortical inhibition and facilitation is modulated by tacs The single-pulse standardized double-stimulation MEP amplitude ratios ± S.E.M. are depicted for ISIs revealing inhibitory (ISIs of 2 and 4 ms) and facilitatory (ISIs of 7, 9 and 12 ms) effects for the different tacs protocols. A and C, 80 Hz(A) and 250 Hz (C) stimulation do not shift inhibition and facilitation relative to the. B, 140 Hz stimulation reduces SICI. D, bar graphs show the mean of MEP value (± S.E.M.) for SICI (ISI 2 and 4 ms) and ICF (ISI 7, 9 and 12 ms) for each condition. P < 0.05, Fisher s LSD test.

10 4900 V. Moliadze and others J Physiol Table 2. Results of the repeated measurement ANOVAs for the study with regard to I-O curve, SICI/ICF, LICI and CSP protocols Factor df F P I-O Type of stimulation Intensity <0.01 Type of stimulation intensity SICI Type of stimulation 3 3, ISI Type of stimulation ISI ICF Type of stimulation ISI Type of stimulation ISI LICI Type of stimulation ISI <0.01 Type of stimulation ISI CSP Type of stimulation Intensity Type of stimulation Intensity Bold indicates significant values ( P < 0.05). TRNS application had no effect on SICI, LICI, CSP or motor-evoked recruitment curves. In the present study, interestingly 140 Hz stimulation reduced SICI. This suggests that the excitability of the interneurones before PST0 PST5 PST10 PST20 PST30 PST40 80 Hz stimulation 140 Hz stimulation 250 Hz stimulation Figure 4. Motor task-related modulation of different frequency tacs The figure shows mean amplitudes of MEPs and their S.E.M. after tac stimulation under motor activation up to 60 min. Stimulation of 80 Hz AC and 250 Hz did not modify the MEP amplitudes significantly, when compared with. In contrast, 140 Hz stimulation during contraction still induced a significant elevation in MEP amplitude compared to. According to the post hoc analysis, significantly increased MEPs were observed in 140 Hz at the PST5 PST50 time points compared to the time point using sham stimulation. Filled symbols indicate significant deviations of the post-measurements of MEP amplitudes from baseline values. P < 0.05, Fisher s LSD test. PST50 PST60 involved is affected by this frequency of stimulation. Pharmacological studies have suggested that SICI at lower conditioning stimulation intensities as used here is mediated through the activation of GABA A receptors (Ziemann et al. 1996; Di Lazzaro et al. 2005; Paulus et al. 2008) whereas LICI is mediated by GABA B receptors (McDonnell et al. 2006). This apparent result seems to be at odds with the classical inhibitory action of GABA, but can be explained by the paradoxical excitatory effects of GABA published in this context recently: in human neocortical slices high frequency stimulation with 130 Hz at 1 ma evoked the release of endogenous GABA via subthreshold activation of voltage-gated sodium channels (Mantovani et al. 2006). More recently, it was shown that the effects of 130 Hz stimulation in this model are mediated by paradoxically facilitatory autoreceptors located on soma, dendrites and axon terminals of GABAergic neurons (Mantovani et al. 2009) well in line with findings of excitatory GABAergic axo-axonic cells (Szabadics et al. 2006). Axo-axonic cells can depolarize pyramidal cells and can initiate stereotyped series of synaptic events in cortical networks because of a depolarized reversal potential for axonal relative to perisomatic GABAergic inputs. As summarized recently, during ripple oscillations basket and bistratified cells increase their firing rate strongly and discharge, whereas in contrast, both axo-axonic and oriens lacunosum-moleculare (O-LM) cells are silenced during and after it, and O-LM cell firing is suppressed during ripples. Since these different interneurons innervate distinct domains of pyramidal cells, they concurrently imprint a spatiotemporal GABAergic conductance matrix onto these cells (Klausberger & Somogyi, 2008). In vivo fast IPSPs on the somata of pyramidal cells have been proposed as a potential mechanism of high-frequency oscillation generation mainly in the hippocampus (Buzsaki et al. 1992; Ylinen et al. 1995). In vitro slice recordings suggest an important synchronizing role for spikelets during spontaneous high-frequency oscillations (Draguhn et al. 1998). Epsztein et al. (2010) have shown that a high incidence of spikelets that occur either in isolation or in bursts could drive spiking as fast as prepotentials of action potentials with the burst firing rate peaking at 138 Hz. A recent study observed that externally induced neuronal plasticity is highly dependent on the physiological state of the subject during stimulation. The average MEP decreases after mental effort and motor activation using tdcs (Antal et al. 2007), paired associative stimulation (PAS) (Stefan et al. 2004), theta burst stimulation (TBS) (Huang et al. 2008b) and trns (Terney et al. 2008). It was suggested that contraction may have changed the membrane potential or Ca 2+ concentration of postsynaptic neurons, and these affected the response to the conditioning protocol (Huang et al. 2008b). Recent

11 J Physiol High frequency oscillations and brain excitability 4901 findings also suggest that Ca 2+ dynamics determine the polarity of LTP/LTD-like changes in vivo (Wankerl et al. 2010). Here we show for the first time that high frequency tacs in the ripple range is also affected by this mechanism. Stimulation at 140 Hz is, however, able to override this inhibition to some extent, underscoring further the particular excitatory efficacy of this frequency. Obviously the 140 Hz stimulation induces more powerful excitatory after-effects than the control conditions, compensating partially and better for the activity induced loss of MEP size than, for example, itbs or anodal tdcs. Another possible explanation of our findings is that the external stimulation of the cortical network leads to modifications in the vascular system that might result in secondary changes in cortical excitability. Indeed, a synchronous activation of large number of neurons may also have metabolic consequences, changing the perineuronal milieu and triggering vascular responses as it has 480 A 80 Hz stimulation 480 B 140 Hz stimulation Reaction Time Reaction Time Blocks Blocks 480 C 250 Hz stimulation 440 Reaction Time Blocks Figure 5. Serial reaction time task (SRTT) and tacs Stimulation at 80 Hz and 140 Hz of the primary motor cortex did not improve implicit motor learning. Interestingly, with 250 Hz a slight but non-significant improvement was evident in Blocks 2 5. Reduced RTs in Blocks 7 and 8 have been identified in two tacs frequency conditions: 80 and 250 Hz, compared to the sham condition. This effect was missing in the 140 Hz tacs condition.

12 4902 V. Moliadze and others J Physiol been documented for rtms (Paus et al. 1998; Pecuchet al. 2000). Because we did not measure metabolic changes, further studies are required to clarify this question. We cannot exclude a minor modification of tacs after-effects by TMS during tacs application. However, so far in general an influence on cortical excitability with very low stimulation frequencies has only been proven when additional measures were introduced such as ischaemic deafferentation (Ziemann et al. 1998). Furthermore, the after-effects seen here are concordant to those observed in many studies using anodal tdcs (overview in: Ziemann et al. 1998; Nitsche et al. 2008) and one concerning trns results (Terney et al. 2008). We are aware of the fact that spontaneous ripple oscillations in the human brain are short-lived in the hippocampus and that the present stimulation protocol only mimics the frequency, not the duration, of ripples in man. Nevertheless here we provide the proof of principle and further studies may be even more efficient when using intermittent ripple stimulation in analogy to, for example, itbs. A similar approach was recently pursued by Groppa et al. (2010) showing that intermittent sinusoidally modulated tdcs provided similar excitatory or inhibitory after-effects when the amplitude was accordingly increased. Transcranial application of high oscillatory frequencies is of great interest in movement disorders as well. Skull thickness and the related distance between the stimulation electrode and cortex is probably not a problem: according to Gabriel et al. (1996) in the frequency range employed here a linear resistance can be assumed for the tissues between the skin and the brain without an attenuation of the effects of higher frequencies. From a therapeutic viewpoint, our findings might lead to an implementation of tacs as a therapeutic tool, e.g. positively influencing cognition and motor performance of patients with aphasia, neglect or dementia, as has already been seen using repetitive TMS (for a review see Thut & Miniussi, 2009). The results represent a further step to more targeted plasticity modulating protocols and also may have important implications for psychiatric diseases accompanied by pathological oscillations. A main difference from deep brain stimulation is the type of current used. Whereas tacs stimulates with sinusoidal electric fields, deep brain stimulation uses short pulses, which according to Gradinaru et al. (2009) works via direct stimulation of afferent axons projecting to the subthalamic nucleus. To our knowledge it is unclear if sinusoidal deep brain stimulation in this frequency range is able to induce spikes in fibre tracts. Just as assumed for tdcs, it may execute its effects via modulation of nerve membranes instead of inducing spikes. An interesting control experiment would be applying rtms with 80, 140 and 250 Hz. Quite apart from the safety concerns, to our knowledge no stimulators are available with this high repetition rate. Also it will be interesting to investigate the effects of even higher tacs frequencies in future. Some of the results of our study may be vulnerable to Type II statistical error. Therefore additional studies with larger sample sizes, focusing on as yet non-significant results presented here are warranted. In summary, the modification of cortical activity through the application of high-frequency transcranial oscillations may adjust behaviourally maladaptive brain states and induce a new balance, pushing the network toward restoring adequate synchronisation and excitation. References Abbruzzese G & Trompetto C (2002). Clinical and research methods for evaluating cortical excitability. JClin Neurophysiol 19, Amassian VE & Stewart M (2003). Motor cortical and other cortical interneuronal networks that generate very high frequency waves. Suppl Clin Neurophysiol 56, Antal A, Terney D, Poreisz C & Paulus W (2007). Towards unravelling task-related modulations of neuroplastic changes inducedinthehumanmotorcortex.eur J Neurosci 26, Bragin A, Engel J Jr, Wilson CL, Fried I & Buzsaki G (1999). High-frequency oscillations in human brain. Hippocampus 9, Buzsaki G, Horvath Z, Urioste R, Hetke J & Wise K (1992). High-frequency network oscillation in the hippocampus. Science 256, Chen R (2000). Studies of human motor physiology with transcranial magnetic stimulation. Muscle Nerve Suppl 9, S Deans JK, Powell AD & Jefferys JG (2007). Sensitivity of coherent oscillations in rat hippocampus to AC electric fields. JPhysiol583, Di Lazzaro V, Oliviero A, Saturno E, Dileone M, Pilato F, Nardone R, Ranieri F, Musumeci G, Fiorilla T & Tonali P (2005). Effects of lorazepam on short latency afferent inhibition and short latency intracortical inhibition in humans. JPhysiol564, Diba K & Buzsaki G (2007). Forward and reverse hippocampal place-cell sequences during ripples. Nat Neurosci 10, Draguhn A, Traub RD, Schmitz D & Jefferys JG (1998). Electrical coupling underlies high-frequency oscillations in the hippocampus in vitro. Nature 394, Engel J Jr, Bragin A, Staba R & Mody I (2009). High-frequency oscillations: what is normal and what is not? Epilepsia 50, Epsztein J, Lee AK, Chorev E & Brecht M (2010). Impact of spikelets on hippocampal CA1 pyramidal cell activity during spatial exploration. Science 327, Fuentes R, Petersson P, Siesser WB, Caron MG & Nicolelis MA (2009). Spinal cord stimulation restores locomotion in animal models of Parkinson s disease. Science 323,

13 J Physiol High frequency oscillations and brain excitability 4903 Fuhr P, Agostino R & Hallett M (1991). Spinal motor neuron excitability during the silent period after cortical stimulation. Electroencephalogr Clin Neurophysiol 81, Gabriel S, Lau RW & Gabriel C (1996). The dielectric properties of biological tissues: II. Measurements in the frequency range 10 Hz to 20 GHz. Phys Med Biol 41, Gradinaru V, Mogri M, Thompson KR, Henderson JM & Deisseroth K (2009). Optical deconstruction of parkinsonian neural circuitry. Science 324, Grenier F, Timofeev I & Steriade M (2001). Focal synchronization of ripples ( Hz) in neocortex and their neuronal correlates. J Neurophysiol 86, Grenier F, Timofeev I & Steriade M (2003). Neocortical very fast oscillations (ripples, Hz) during seizures: intracellular correlates. J Neurophysiol 89, Groppa S, Bergmann TO, Siems C, Molle M, Marshall L & Siebner HR (2010). Slow-oscillatory transcranial direct current stimulation can induce bidirectional shifts in motor cortical excitability in awake humans. Neuroscience 166, Hammond C, Bergman H & Brown P (2007). Pathological synchronization in Parkinson s disease: networks, models and treatments. Trends Neurosci 30, Huang YZ, Rothwell JC, Edwards MJ & Chen RS (2008a). Effect of physiological activity on an NMDA-dependent form of cortical plasticity in human. Cereb Cortex 18, Huang YZ, Rothwell JC, Edwards MJ & Chen RS (2008b). Effect of physiological activity on an NMDA-dependent form of cortical plasticity in human. Cereb Cortex 18, Jacobs J, Levan P, Chatillon CE, Olivier A, Dubeau F & Gotman J (2009). High frequency oscillations in intracranial EEGs mark epileptogenicity rather than lesion type. Brain 132, Kanai R, Chaieb L, Antal A, Walsh V & Paulus W (2008). Frequency-dependent electrical stimulation of the visual cortex. Curr Biol 18, Klausberger T & Somogyi P (2008). Neuronal diversity and temporal dynamics: the unity of hippocampal circuit operations. Science 321, Kujirai T, Caramia MD, Rothwell JC, Day BL, Thompson PD, Ferbert A, Wroe S, Asselman P & Marsden CD (1993). Corticocortical inhibition in human motor cortex. JPhysiol 471, Mantovani M, Moser A, Haas CA, Zentner J & Feuerstein TJ (2009). GABA A autoreceptors enhance GABA release from human neocortex: towards a mechanism for high-frequency stimulation (HFS) in brain? Naunyn Schmiedebergs Arch Pharmacol 380, Mantovani M, Van Velthoven V, Fuellgraf H, Feuerstein TJ & Moser A (2006). Neuronal electrical high frequency stimulation enhances GABA outflow from human neocortical slices. Neurochem Int 49, McDonnell MN, Orekhov Y & Ziemann U (2006). The role of GABA B receptors in intracortical inhibition in the human motor cortex. Exp Brain Res 173, Middleton SJ, Racca C, Cunningham MO, Traub RD, Monyer H, Knopfel T, Schofield IS, Jenkins A & Whittington MA (2008). High-frequency network oscillations in cerebellar cortex. Neuron 58, Nissen MJ & Bullemer P (1987). Attentional requirements of learning: evidence from performance measures. Cognit Psychol 19, Nitsche M, Cohen L, Wassermann E, Priori A, Lang N, Antal A, Paulus W, Hummel F, Boggio P, Fregni F & Pascual-Leone A (2008). Transcranial direct current stimulation: State of the art Brain Stimul 1, Nitsche M & Paulus W (2001). Sustained excitability elevations induced by transcranial DC motor cortex stimulation in humans. Neurology 57, Nitsche MA, Schauenburg A, Lang N, Liebetanz D, Exner C, Paulus W & Tergau F (2003). Facilitation of implicit motor learning by weak transcranial direct current stimulation of the primary motor cortex in the human. JCognNeurosci15, Oldfield R (1971). The assessment and analysis of handedness: the Edinburgh inventory. Neuropsychologia 9, Paulus W, Classen J, Cohen LG, Large CH, Di Lazzaro V, Nitsche M, Pascual-Leone A, Rosenow F, Rothwell JC & Ziemann U (2008). State of the art: Pharmacologic effects on cortical excitability measures tested by transcranial magnetic stimulation. Brain Stimul 1, Paus T, Jech R, Thompson CJ, Comeau R, Peters T & Evans AC (1998). Dose-dependent reduction of cerebral blood flow during rapid-rate transcranial magnetic stimulation of the human sensorimotor cortex. J Neurophysiol 79, Pecuch PW, Evers S, Folkerts HW, Michael N & Arolt V (2000). The cerebral hemodynamics of repetitive transcranial magnetic stimulation. Eur Arch Psychiatry Clin Neurosci 250, Pogosyan A, Gaynor LD, Eusebio A & Brown P (2009). Boosting cortical activity at beta-band frequencies slows movement in humans. Curr Biol 19, Rothwell JC, Hallett M, Berardelli A, Eisen A, Rossini P & Paulus W (1999). Magnetic stimulation: motor evoked potentials. The International Federation of Clinical Neurophysiology. Electroencephalogr Clin Neurophysiol Suppl 52, Schroeder CE & Lakatos P (2009). Low-frequency neuronal oscillations as instruments of sensory selection. Trends Neurosci 32, Singer W (2009). Distributed processing and temporal codes in neuronal networks. Cogn Neurodyn 3, Stefan K, Wycislo M & Classen J (2004). Modulation of associative human motor cortical plasticity by attention. J Neurophysiol 92, Steriade M, McCormick DA & Sejnowski TJ (1993). Thalamocortical oscillations in the sleeping and aroused brain. Science 262, Szabadics J, Varga C, Molnar G, Olah S, Barzo P & Tamas G (2006). Excitatory effect of GABAergic axo-axonic cells in cortical microcircuits. Science 311, Terney D, Chaieb L, Moliadze V, Antal A & Paulus W (2008). Increasing human brain excitability by transcranial high-frequency random noise stimulation. JNeurosci28, Thut G & Miniussi C (2009). New insights into rhythmic brain activity from TMS-EEG studies. Trends Cogn Sci 13,