Speech Dynamics Are Coded in the Left Motor Cortex in Fluent Speakers But

Brain Advance Access published January 15, 2015 doi:10.1093/brain/awu390 BRAIN 2015: Page 1 of 14 | 1

Speech dynamics are coded in the left motor cortex in ﬂuent speakers but not in adults who stutter

Nicole E. Neef,1,2,Ã T. N. Linh Hoang,1,Ã Andreas Neef,3 Walter Paulus1 and Martin Sommer1

*These authors contributed equally to this work.

The precise excitability regulation of neuronal circuits in the primary motor cortex is central to the successful and fluent production of speech. Our question was whether the involuntary execution of undesirable movements, e.g. stuttering, is linked to an insufficient excitability tuning of neural populations in the orofacial region of the primary motor cortex. We determined the speech- related time course of excitability modulation in the left and right primary motor tongue representation. Thirteen fluent speakers Downloaded from (four females, nine males; aged 23–44) and 13 adults who stutter (four females, nine males, aged 21–55) were asked to build verbs with the verbal prefix ‘auf’. Single-pulse transcranial magnetic stimulation was applied over the primary motor cortex during the transition phase between a fixed labiodental articulatory configuration and immediately following articulatory configurations, at by guest on January 18, 2015 different latencies after transition onset. Bilateral electromyography was recorded from self-adhesive electrodes placed on the surface of the tongue. Off-line, we extracted the motor evoked potential amplitudes and normalized these amplitudes to the individual baseline excitability during the fixed configuration. Fluent speakers demonstrated a prominent left hemisphere increase of motor cortex excitability in the transition phase (P=0.009). In contrast, the excitability of the right primary motor tongue representation was unchanged. Interestingly, adults afflicted with stuttering revealed a lack of left-hemisphere facilitation. Moreover, the magnitude of facilitation was negatively correlated with stuttering frequency. Although orofacial midline muscles are bilaterally innervated from corticobulbar projections of both hemispheres, our results indicate that speech motor plans are controlled primarily in the left primary speech motor cortex. This speech motor planning-related asymmetry towards the left orofacial motor cortex is missing in stuttering. Moreover, a negative correlation between the amount of facilitation and stuttering severity suggests that we discovered a main physiological principle of fluent speech production and its role in stuttering.

1 Department of Clinical Neurophysiology, Georg-August-University, Go¨ ttingen, Germany 2 Max Planck Institute for Human Cognitive and Brain Sciences, Department of Neuropsychology, Leipzig, Germany 3 Max Planck Institute for Dynamics and Self-Organization, Department of Nonlinear Dynamics, Go¨ ttingen, Germany Correspondence to: Nicole E. Neef, PhD, Department of Clinical Neurophysiology, University of Go¨ ttingen Robert-Koch-Str. 40, D – 37075 Go¨ ttingen E-mail: [email protected]

Keywords: motor control; primary motor cortex; transcranial magnetic stimulation; motor evoked potentials, stuttering Abbreviations: BA = Brodmann area; MEP = motor evoked potential; TMS = transcranial magnetic stimulation

Received August 1, 2014. Revised October 17, 2014. Accepted November 17, 2014. ß The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected] 2 | BRAIN 2015: Page 2 of 14 N. E. Neef et al.

cannot be employed in humans. Neuroimaging studies Introduction have shown aberrant activity in the primary motor cortex Effortless and fluent speech production is essential for suc- during stuttering, which has been taken as an indirect sign cess in society. Persistent stuttering is a neurodevelopmental of deviant excitability, and this has been interpreted in speech fluency disorder with a complex genetic basis favour of an imbalance of excitability (Ludlow and (Fisher, 2010). It occurs in 5% of all children and persists Loucks, 2003). in about 1% of the adult population (Yairi and Ambrose, Transcranial magnetic stimulation (TMS) is a non-inva- 2005), severely compromising quality of life (Yaruss, sive technique that reads out motor cortex excitability by 2010). stimulating a subset of neurons in a small part—approxi- 3 MRI provides evidence that neural structures and neural mately 1 cm —of the cerebral cortex (Salvador et al., activity exhibit irregularities in persons with persistent de- 2011). The technique uses the principle of electromagnetic velopmental stuttering. The main findings are an imbal- induction. A very brief magnetic pulse induces an electrical anced activation of speech-related auditory and motor field, and thereby an electrical current, in conductive brain cortices (Brown et al., 2005), a reduced activation of sub- tissue. Strong, suprathreshold stimuli applied over the cortical structures, including the basal ganglia and cerebel- motor cortex directly elicit motor evoked potentials lum (Giraud et al., 2008; Watkins et al., 2008), a reduced (MEPs) in the target muscles of the stimulated area. The white matter integrity of left hemispheric speech motor re- MEP peak-to-peak amplitude is a common quantifier of gions (Sommer et al., 2002; Watkins et al., 2008; Kell cortical excitability. et al., 2009; Cai et al., 2014), and a left inferior frontal- Previous TMS examinations of adults who stutter focused on the corticospinal excitability of the primary premotor functional-connectivity deficit (Chang et al., motor hand representation (Sommer et al., 2009; Alm 2011; Chang and Zhu, 2013). For reports describing the et al., 2013; Busan et al., 2013). Studies of the motor variability across previous imaging investigations see Cai cortex excitability for orofacial structures with TMS are et al. (2014), Ingham et al. (2012) and Nil et al. (2008).

scarce and challenging (Devlin and Watkins, 2007), only Downloaded from It is plausible to assume that this irregular structural and a single study examined the speech motor cortex excitabil- functional connectivity relates to an irregular up- or down- ity in adults who stutter (Neef et al., 2011b). In that study, regulation of the local circuitry, or local excitability. we measured MEP responses at rest and at different inter-

Regulation of excitability is the mechanism by which by guest on January 18, 2015 vals after an artificial stimulus, i.e. a conditioning TMS motor programs are organized, selected, and finally exe- pulse. In adults who stutter, cortical excitability was modu- cuted to perform a coordinated movement (Stinear et al., lated less compared to fluent speakers. To elucidate 2009). Precise regulation of the excitability of neuronal cir- whether this reduced dynamic range of excitability modu- cuits in the primary motor cortex is central to the successful lation also occurs during speech production, ultimately, execution of speech movements. These neurons receive TMS pulses have to be applied during speaking. To moni- input from a large distributed network and, as neurons of tor the modulation of excitability in response to a certain the final motor-output stage, integrate this input to com- change in cortical activity, we replaced the artificial condi- mand coordinated, voluntary movements. Theories suggest tioning TMS stimulus by speech-driven, intrinsic modula- that the executed motor program is the ‘winner’ selected tory activity. Across trials, the initial excitability state was out of a number of competing, simultaneously-activated controlled for cognitive load and myoactivity, as subjects neural representations of potential actions (Cisek and prolonged a fixed articulatory configuration and prepared Kalaska, 2005). The accidental execution of an undesirable the pronunciation of a word chosen from a linguistically movement, e.g. a stutter event, might therefore be triggered homogeneous set. Consequently, in the current study, we by a failure in motor program activation which, on the provide the first online assessment of motor cortex excit- physiological level, is reflected in a dynamic imbalance be- ability of the tongue representation during speaking. We tween the facilitation and inhibition of neural populations show that speech production fails to produce an increase in the primary motor cortex (Michelet et al., 2010). Thus, in speech-specific left motor cortex excitability in adults intracortical inhibitory and excitatory networks of the pri- who stutter, which contrasts to the situation in fluent mary motor cortex, which are driven by large amounts of speakers. input information from the cortical and subcortical regions, control the selection and initiation of target movements and suppress undesirable synergism (Stinear et al., 2009). This theory clearly predicts a deviant excitability of the primary Materials and methods motor cortex in stuttering which should be displayed in altered patterns of speech-induced inhibition and facilita- Participants tion. This prediction is notoriously hard to address because A total of 33 adults were recruited. All were native speakers of no animal models exist to investigate speech motor control German. Data on 13 adults who stutter (four females; mean apart from birdsong (Wang et al., 2008), and invasive 34.5 years, SD = 12.0) and 13 fluent speakers (four females, methods which could directly measure these effects mean 30.1 years, SD=7.8) were included in the analysis. We M1 tongue area excitation during speech BRAIN 2015: Page 3 of 14 | 3 excluded three subjects because 140% of their motor threshold mounted on a customized spoon-shaped silicon mouthpiece. was 480% of the maximum stimulator output, three other Contact area at the tongue was 5 mm Â 10 mm at longitudinal subjects because they perceived the set-up as uncomfortable and lateral inter-electrode distances of 25 and 20 mm, respect- and refused further participation, and one adult in the group ively. The mouthpiece was placed on the upper surface of the of fluent speakers was excluded because he showed 5% stut- tongue, and the subjects were asked to close their lips and tered syllables on speech analysis. Adults who stutter were teeth comfortably without additional pressure and to hold recruited from the local stuttering support group and from the end of the mouthpiece with the hand ipsilateral to the the Institute for Kassel Stuttering Therapy (Euler et al., TMS stimulation site, with the elbow comfortably supported. 2009). Fluent speakers were recruited by advertisement. The During the recordings, participants were asked to press their groups were matched for age, handedness (Oldfield, 1971), tongue slightly against the electrodes and their lower teeth. and education (1 = school; 2 = high school; 3 = 52 years col- This procedure was described previously (Neef et al., 2011b). lege; 4 = 2 years college; 5 = 4 years college; 6 = postgraduate). Surface electromyographic signals were amplified (Â1000) and Eight adults who stutter reported a family history of stuttering. Butterworth bandpass-filtered (bandwidth 20 Hz to 2 kHz) None of the fluent speakers reported having a family history of with a Digitimer D360 amplifier, acquired at a sampling fre- speech or language disorders. Apart from stuttering in the quency of 5 kHz, using a 1401 laboratory interface group of adults who stuttered, participants reported no med- (Cambridge Electronic Design). Recordings were controlled ical history, neurological impairment, or drug use that would by Signal Software (Cambridge Electronic Design, v 2.13) potentially affect their neurological function. Before experi- and stored on a personal computer. An additional channel of mental measures were obtained with TMS, all subjects were the system served to record audio signals of the elicited speech screened for exclusion criteria using a standard TMS safety simultaneous with the electromyographic signals. To do so, we screen (Keel et al., 2001). All subjects provided written in- attached a wireless microphone (AKG PT 40) to the mouth- formed consent prior to inclusion in the study. All procedures piece and fed the acquired audio signal into a third channel of used in this study were approved by the Institutional Review the A/D converter. Board of the University Medical Centre Go¨ ttingen. Stuttering severity was assessed by collecting samples of reading aloud and spontaneous speech elicited through a stan- Transcranial magnetic stimulation Downloaded from dardized interview asking participants to narrate their daily Single-pulse TMS of the primary motor cortex was delivered routine, to retell their favourite movie or novella and to give using a Magstim 2002 magnetic stimulator with a monophasic directions when imagining a person asking the way. These current waveform (Magstim Company). The magnetic stimu- samples were video-recorded and analysed offline by a quali- lator was connected to a standard figure-of-eight coil with a by guest on January 18, 2015 fied speech–language pathologist. The stuttering severity index mean loop diameter of 7 cm. The intersection of the coil was (SSI-3) was used to determine the frequency and duration of held tangentially to the skull with the handle pointing back- stuttered syllables as well as physical concomitants of stutter- wards and laterally at an angle of 45 to the sagittal plane in ing. Stuttering severity in the adults who stutter was as fol- order to generate a posterior-anterior current in the brain lows: six were very mild, one was mild, two were moderate, (Kaneko et al., 1996; Di Lazzaro et al., 2004). The optimal three were severe, and one was very severe. Table 1 summar- position of the coil was defined as the site where stimulation izes the demographic information of the participants. consistently resulted in the largest MEPs. For localization, the scalp surface was explored systematically, the position for con- Electromyography sistently inducing maximal MEPs in the contralateral tongue- site at the lowest stimulus strength was identified as the ‘hot Surface recordings of the lingual muscle were made from the spot’ and marked with a pen to ensure accurate coil placement contralateral and ipsilateral tongue-side with two pairs of dis- throughout the experiment (Muellbacher et al., 2001; Neef posable, pre-gelled, silver/silver chloride ring electrodes et al., 2011b). The interstimulus interval between single TMS (5 mm Â 100 mm, Viasys Neurocare). Electrodes were pulses was 6 s (Æ10%, 0.2 Hz). A maximum of 50 pulses

Table 1 Participants demographic information, behavioural results and TMS motor thresholds

Measures Stuttering Controls Difference n 13 13 n/a Females (n) 4 4 n/a Age in years 34.5 (7.8) 30.2 (12.0) P=0.3 (n.s.) Age of stuttering onset in years 4.0 (2.1) n/a n/a SSI-3 overall score 23.5 (8.9) n/a n/a % Stuttered disﬂuencies 7.6 (6.0) 0.3 (0.2) P=0.001 Handedness 75.2 (43.6) 87.6 (17.8) P=0.35 (n.s.) Education* 3 4 P=0.09 (n.s.) Motor threshold left hemisphere 41.9 (7.3) 37.6 (3.2) P=0.07 (n.s.) Motor threshold right hemisphere 42.6 (5.6) 37.7 (3.4) P=0.01

*Education is reported as median, group differences were quantified by Mann-Whitney test. SSI-3 = Stuttering Severity Instrument, third edition; % stuttered disfluencies = stuttered syllables occurring per 100 syllables; n.s. = not significant. 4 | BRAIN 2015: Page 4 of 14 N. E. Neef et al. was applied before replacing the self-adhesive electrodes. The lasted 5 min. A single TMS pulse was delivered per trial, frequent change of the electrodes was necessary because of with an intertrial interval of 12 s. Motor cortex excitability salivation. It took 5 min to clean the mouth piece, precisely was measured at seven different time points: (i) during the mount the new electrodes and prepare the subject for the sub- resting state, 2900 ms after the trial started; (ii) while holding sequent measurements. A maximum of 100 pulses was applied the fixed articulatory configuration of the labio-dental fricative to determine the hotspot and the motor threshold. Because of [f] 1000 ms after the first go-signal; and (iii) at five different the low rate of pulse repetition we ruled out a modulation of latencies of the transition phase between the fixed articulatory motor cortex excitability by TMS pulses applied during this configuration and the target speech gestures, namely 240 ms, procedure (Fitzgerald et al., 2006). The hot spot of the motor 280 ms, 320 ms, 360 ms, and 400 ms after the second go- tongue area was 2–3 cm anterior and 1–2 cm lateral to the signal. A total of 14 MEPs were acquired per condition. hand representation, which is consistent with the literature Importantly, the baseline, resting state MEP measurement (Svensson et al., 2003). Single TMS pulses were applied to occurs before the participants had been asked to read the determine the minimal stimulus intensity to the nearest 1% word and to keep it in their mind directly before probing ex- of the maximum stimulator output required to produce citability. Had they already read the word the additional cog- MEPs of 4100 mV in at least three of six consecutive stimuli. nitive load and the speech preparatory behaviour might engage This intensity defines the motor threshold and was set to different neuronal processes and thus result in incomparable 120% throughout the experiment. dynamic states of the speech motor cortex. This is suggested To check that there is a sufficient dynamic range of excit- by a previous MEG study reporting a reversed cortico-cortical ability modulation and ceiling effects due to saturation are not processing chain for delayed single-word reading (Salmelin present, we acquired MEP input-output curves. This procedure et al., 2000). was adopted from Ro¨ del et al. (2003). We elicited MEPs by stimulating stepwise during 30 trials, rising from 90% to 100%, 110%, 120%, 130%, and 140% of motor threshold. Data analysis Data analyses were performed using a custom-written EMG- Verbal stimuli and speech task Browser in Igor Pro (Wavemetrics). Acoustic speech waveforms were visually inspected in the data browser and speech Downloaded from Verbal stimuli consisted of 49 German verbs always starting onset time was manually determined at the time point where with the prefix ‘auf’ and always continuing with a consonantal the waveform moved away from the baseline at the first clear cluster, e.g. ‘aufbleiben’ (to stay up). Words were selected from pulse of the vocal folds. We visually examined the electromyo-

the database Webcelex (MPI Nijmegen, NL; celex.mpi.nl) and graphic signal of all recordings, manually segmented all valid by guest on January 18, 2015 controlled for the linguistic parameters frequency, phonetic MEPs, and excluded signals with TMS artefacts lasting beyond complexity, number of letters, number of phonemes, number the motor-evoked response. All valid recordings were fed into of syllables, and word accent. consecutive analyses. To determine pre-TMS tongue activity Figure 1A illustrates the time course of a single trial of the 200 ms of the EMG signal immediately before the TMS arte- speech task. Each trial started with a blank interval of 4000 ms fact were corrected for offset, rectified and averaged over the followed by a 1500 ms presentation of a verb without its cor- two electrode pairs attached to the left side of the tongue and responding prefix. Subjects were instructed to read the verb to the right side of the tongue. silently and to remember it. Another blank interval of Mean peak-to-peak amplitudes were extracted separately for 3000 ms was followed by a plus sign informing the participant each condition, for the contralateral and ipsilateral projection to speak the prefix ‘auf’ and to prolong its labio-dental frica- in the left and the right hemisphere. To determine the tive [f]. After 1500 ms a question mark appeared prompting response-locked time course of excitability modulation, we ex- the articulation of the verb that the participant had previously tracted acoustic latencies relative to the individual speech onset read. The whole sequence was repeated after 12 s. of the target. MEP latencies were calculated setting the target The insertion of the prefix ‘auf’ was planned to force the speech onset as time zero. The selection of 50 ms bins, starting cortical dynamics to a predefined state. We wanted to ensure 40 ms after word onset. No less than nine observations per bin (i) a comparable cognitive load (keep a word in mind); (ii) a were present for each time bin in each group. For later statis- comparable myogenic state (prolonging a labiodental fricative); tical analyses we performed normalization in two cases. First, and (iii) a comparable preparation of a motor response MEP amplitudes during the prolongation were related to MEP (waiting for a go signal). amplitudes during rest. Grand averages are plotted in Fig. 3. Second, MEP amplitudes of the transition phase were related to the absolute MEP amplitude during prolongation Experimental design Fig. 4B. All absolute grand average MEP amplitudes are Participants were tested on both hemispheres, in two different plotted in Fig. 4A. TMS sessions, separated by at least 48 h. The order of the tested hemispheres was balanced between subjects. In each ses- Statistical analyses sion, we began with baseline assessments of the resting motor threshold and input-output curve. This was followed by a fa- All statistical analyses were performed using SPSS (IBM SPSS, miliarization period where the participants were asked to per- Statistics for Windows, Version 22.0). Data of all metric vari- form 10 trials of the speech task with the mouthpiece in the ables were first fed into Kolmogorov-Smirnov tests for each correct position but without the delivery of TMS pulses. In the group separately to evaluate whether the samples come from core experiment, 98 trials were split into two blocks of 35 a population with a specific distribution. Group differences of trials and one block of 28 trials. Pauses between blocks age, handedness, percentage of stuttered syllables, and motor M1 tongue area excitation during speech BRAIN 2015: Page 5 of 14 | 5 Downloaded from by guest on January 18, 2015

Figure 1 Experimental set-up and procedure. (A) Flowchart of trial. Participants silently read a German verb presented on a screen. A ﬁrst visual go-signal ( + ) instructed the participant to start speaking the invariant preﬁx ‘auf’ and to prolong the ‘fff’ until a second visual go-signal (!) indicated to move onto speaking the previously presented verb. To study a time window of 350 ms preceding the second speech gesture, we used 70 single pulse TMS trials per hemisphere per subject, in which we varied the interval between the single TMS pulse and the speech onset of the verb stem. (B) Traces of the electromyography with MEPs elicited in the contralateral (right) and ipsilateral (left) tongue-side by TMS of the left primary motor cortex. Stimulation intensity was 120% of the individual’s motor threshold. The lower line shows the oscillogram indicating speech onset of the particle ‘auf’ and the subsequent speech signal.

thresholds were tested by two-tailed unpaired t-tests. Group Hemisphere (left, right), and Tongue projection (ipsilateral, differences of education, speech onset times, and pre-TMS contralateral) as within-subject factor. tongue activity at baseline and during prolongation were To test for group differences in speech-specific modulation of tested by Mann-Whitney tests. motor cortex excitability, we used two-tailed unpaired t-tests To test the variance of pre-TMS tongue activity in the and thus compared the normalized MEP amplitudes (normal- transition phase a mixed-model ANOVA was calculated ized to the augmented baseline excitability, while holding the with Hemisphere (left, right) and Time (À50 ms, À100 ms, fixed articulatory configuration of the labio-dental fricative). À150 ms, À200 ms) as within-subjects factors and Group as Paired two-tailed t-tests were used to evaluate differences be- between-subjects factor. tween speech-related excitability and baseline excitability. Not To test the variance of motor cortex excitability during pro- all bins contained data from all subjects due to the somewhat longing the German prefix ‘auf’ we employed a mixed-model random delay between go signal and speech onset (Fig. 4B). ANOVA with Group as between-subject factor and The t-tests were performed for eight time bins. Because data in 6 | BRAIN 2015: Page 6 of 14 N. E. Neef et al. the bins are not independent, because of the inherently limited Figure 2A and B illustrate that adults who stutter were as speed with which excitability is modulated, it is not clear to fast (speech onset time = 480 ms) as fluent speakers (speech which degree a correction for multiple comparisons is needed; onset time = 461 ms, U = 92, P=0.724). This implies a uncorrected values are reported. minimal amount of stuttering events throughout the experi- For the group of adults who stutter, we related the mean ment. Figure 2B demonstrates speech onset times of all normalized MEP amplitude across all time bins with the per- trials normalized to the timing of the TMS pulse. Adults centage of syllables stuttered using an exponential function. This was done separately for the two hemispheres and their who stutter show a longer tail at the left illustrating that merged projections. slightly more trials were characterized by longer speech A mixed-model ANOVA was used to test the variance of onset times and maybe stuttering events but those trials motor thresholds with Group (adults who stutter, fluent speak- were not included in the statistical analysis. Respondents ers) as between-subject factor and Hemisphere (left, right) as reported that they had not stuttered during the experiment within-subject factor. Variance in the MEP input-output curves when asked. was tested with a mixed-model ANOVA with Group as be- The number of trials in which the subject started voicing tween-subject factor and Hemisphere, Tongue projection (ipsi- before the TMS pulse was applied is 334 for control sub- lateral, contralateral), and Intensity (90, 100, 110, 120, 130, jects and 336 for adults who stutter (Fig. 2C). 140% motor threshold) as within-subject factors. To control whether speaking with the mouthpiece in place resulted in a change of the motor cortex excitability we com- Adults who stutter show the same pared MEP amplitudes recorded before and after familiarization. The before familiarization condition was characterized by level of the pre-TMS tongue the input-output curve MEPs at 120% motor threshold. innervation as fluent speakers Responses recorded during resting state within a trial served Figure 2D–F depicts tongue activity before TMS. During as after-familiarization MEPs. These MEPs were elicited with the same stimulation intensity, 2900 ms after trial onset, but the within-task resting state condition (2700–2895 ms) before word presentation. We conducted a mixed-model tongue pressure was similar between groups (left hemi- Downloaded from ANOVA with Hemisphere (left, right) and Task (within trial, sphere grand median = 16 mV, P 4 0.05; right hemisphere input-output curve MEP at 120% motor threshold) as within- grand median = 19 mV, P 4 0.05). The same holds true subjects factors and Group as between-subjects factor. We for the prolongation of the voiceless, labio-dental fricative compared motor cortex excitability at the beginning of the (9300–9495 ms, left hemisphere grand median = 30 mV, by guest on January 18, 2015 test phase (first 20 MEPs) with motor cortex excitability at P 4 0.05). Right hemisphere projections show a higher the end of the test phase (last 20 MEP) to exhaustedly test median in fluent speakers (56 mV) compared to adults whether speaking with the mouthpiece affects excitability of who stutter (25 mV) with a grand median = 34 mV, but no the tongue motor cortex over the whole time course of the significant group difference (P 4 0.05). Kolmogorov- experiment. Only MEPs of the transition phase were con- Smirnov tests reject the hypothesis of normal distribution sidered, i.e. the data that entered Fig. 4A and B, but here, MEPs were pooled within subjects regardless of the exact of pre-TMS tongue activity during resting state and during time point when stimulation occurred in the transition phase. the prolongation of the fricative. Therefore, all comparisons We conducted another mixed-model ANOVA with were calculated with independent samples median tests. For Hemisphere (left, right) and Time (first 20 MEPs, last the transition phase after the second go-signal the time 20 MEPs) as within-subjects factors and Group as between- course of tongue activity divided into 50 ms bins over a subjects factor. period of 200 ms. In this case Kolmogorov-Smirnov tests Significant effects in ANOVAs were further evaluated with indicate normality of pre-TMS tongue activity for all con- ANOVAS or t-test. ditions. A mixed model ANOVA yielded no effect of Group, no effect of Hemisphere, and no interactions, but an effect of Time [F(3,22) = 7.85, P=0.006, Greenhouse- Results Geisser corrected]. To summarize, tongue pressure was comparable between groups across conditions and rose Adults who stutter were fluent and as slightly towards the transition phase. fast as fluent speakers when responding to the second go-signal Adults who stutter exhibit a left-lateralized increase of excitability Intelligibility of the utterances was limited due to the mouthpiece. As a consequence, an analysis of the frequency To test the level of excitation during speaking, we normal- of stuttering events in the phrases uttered during the ex- ized MEP amplitudes to resting state activity. While fluent periment would not be reliable. Nevertheless, we can use speakers showed a right-lateralized increase of excitability, the speech onset time distribution to draw conclusions on adults who stutter showed a left-lateralized increase of ex- the performance of the speakers because most disfluencies citability (Fig. 3). This is evident in the interaction occur at word initial position (Bloodstein and Ratner, of Group Â Hemisphere [F(24,1) = 5.0, P=0.036, 2008) and hence would cause longer speech onset times. Greenhouse-Geisser corrected]. Post hoc two-tailed paired M1 tongue area excitation during speech BRAIN 2015: Page 7 of 14 | 7

Trials with SOT A 0.9 BC 500 speaking after TMS speaking before TMS before TMS 140 0.8 time window used for analysis 400 120 0.7 100 0.6 300 80 0.5 200 60 frequency frequency 0.4 Fluent speakers 40 Adults who stutter 100 0.3 20 SOT relative to Go-signal 2 (s) 0.2 0 0 Adults Fluent who speakers 0.8 0.6 0.4 0.2 0.0 -0.2 -0.4 54321 stutter time points delay t SOT - tstim (s)

DE200 F 200 ms pre TMS 200 ms pre TMS 200 ms pre TMS within task within task 50 within task transition phase resting state prolongation [f] 150 40

100 20

tongue activity (µV) 10

tongue activity (µV) 50 0 -200 -150 -50-100 time (ms)

Fluent speakers 0 Adults who stutter left right left right left right left right All subjects hemisphere hemisphere

Figure 2 No group differences for speech onset time and tongue’s pre-TMS innervation. Speech onset times of participants. (A) Downloaded from The medians of the speech onset times are plotted for every subject. A Mann-Whitney test detected no differences between groups (P=0.724). The solid lines represent the group medians. (B) Histograms illustrate the distribution of speech onset time normalized to the time points of the TMS pulse. Bins that appear at the left side of line zero contain the number of trials where the TMS pulse was applied before speech onset. by guest on January 18, 2015 (C) Amount of trials in which TMS occurred after speech onset as shown on the right side of line zero in the histogram. Time points 1 to 5 represent the times of the TMS pulse with regard to the second go-signal (240 ms, 280 ms, 320 ms, 360 ms and 400 ms). (D and E) Tongue activity quantiﬁed as the average over the offset corrected, rectiﬁed EMG signal during a 200 ms interval prior to transcranial magnetic stimulation. EMG signals were averaged over the two electrode pairs attached to the left side of the tongue and to the right side of the tongue. (C) For the transition phase after the second go-signal the time course of tongue activity is separated into 50 ms bins over a period of 200 ms. Tongue pressure was comparable between groups across conditions and rose slightly towards the transition phase. SOT = speech onset time.

t-tests revealed a marginally increased excitation towards normalized MEP amplitudes were significantly increased the right hemisphere in fluent speakers (t = 2.06, P= up to 260 ms before speech onset of the target speech 0.062) and a significantly increased excitation towards movements (0.009 5 P 5 0.049, uncorrected; for all P- the left hemisphere in adults who stutter (t = 2.43, values see Supplementary Table 1). In contrast, right hemi- P=0.032). sphere projections did not show this facilitation in the transition phase to the subsequent speech gestures, except for Fluent speakers have a left-lateralized the ipsilateral projection 110 ms to 60 ms before speech onset (P=0.039, uncorrected). Adults who stutter lacked facilitation of the primary motor a facilitation of neurons in the left hemisphere primary tongue representation before the motor tongue representation (for the P-values see speech segment is executed Supplementary Table 1). To check for differences between adults who stutter and fluent speakers, we ran two-tailed To assess the modulation of excitability in the transition unpaired t-tests. Fluent speakers showed a significantly phase from a fixed articulatory configuration towards increased excitation in the contralateral left hemisphere target speech movements, we normalized MEP amplitudes projection up to À160 ms before speech onset at stimulus-locked time points in the transition phase in (0.004 5 P 5 0.041 uncorrected; for all P-values see relation to the excitation level during the prolongation of Supplementary Table 2). the fricative. Only fluent speakers showed an additional Speaking with the mouthpiece affected excitability of the facilitation of neurons in the left hemisphere primary tongue motor cortex over the whole time course of the motor tongue representation (Fig. 4B). Response-locked experiment. This is the result of the comparison of non- 8 | BRAIN 2015: Page 8 of 14 N. E. Neef et al.

5 Contralateral projection 5 Ipsilateral projection Adults who stutter 4 4 Fluent speakers

3 3

2 2 to resting state to resting state 1 1 MEP amplitude normalized MEP MEP amplitude normalized MEP 0 0 left hemisphere right hemisphere left hemisphere right hemisphere

Figure 3 Prolongation induced left hemisphere facilitation in adults who stutter. Box plots illustrate tongue motor cortex excitability during the prolongation of the labio-dental fricative related to resting state. Normalized MEPs are depicted separately for the contralateral and for the ipsilateral tongue projections (Æ SEM). ANOVA yielded a significant interaction of Group Â Hemisphere for the contralateral projections. Post hoc t-tests resulted in a right-lateralized facilitation in fluent speakers and a left-lateralized facilitation in adults who stutter (filled box). A group differences was significant in the left hemisphere contralateral projection. Filled box indicates significant change (P 5 0.05). Asterisk indicates level of difference between groups: *P 5 0.05.

A 3 Left hemisphere right tongue B 3 Left hemisphere right tongue ** 2 2 ** * *

1 1 Downloaded from

0 0 3 3 Left hemisphere left tongue Left hemisphere left tongue by guest on January 18, 2015

2 2

1 1

0 0

3 Right hemisphere left tongue 3 Right hemisphere left tongue

2 2 absolute MEP amplitude (mV) 1 1

0 0 # subjects 9 3 Right hemisphere right tongue 3 Right hemisphere right tongue 10 11 MEP amplitude normalized to fixed articulatory configuration (BA2) 2 2 12 13 1 1 Fluent speakers 0 0 Adults who stutter 600BA1 BA2 200 0 -300 -200 -100 0 time before speech onset (ms) time before speech onset (ms)

Figure 4 Speech preparation facilitated left motor tongue area in fluent speakers only.(A) Group averages of the absolute MEP amplitudes are plotted for resting state excitability (BA1), excitability during prolonged speaking (BA2) and for the trajectory of excitability towards the target gesture. (B) Normalized excitability of the primary motor tongue representation in the transition phase. The x-axis depicts the reaction time of the transition phase between a fixed articulatory configuration and a target articulatory gesture. Towards the target gesture, excitability of the left motor cortex increases significantly in fluent speakers, whereas adults who stutter do not show such a modulation. Filled symbols indicate significant differences from baseline activity. Error bars indicate the standard error of mean. Asterisks indicate differences between groups: *P 5 0.05; **P 5 0.001. The size of the symbols indicates the number of subjects contributing to the bin. M1 tongue area excitation during speech BRAIN 2015: Page 9 of 14 | 9

A Effect of task B Left hemisphere Right hemisphere C Motor threshold 50 2.0 2.0 * 40 1.5 *** 1.5 30 20 1.0 1.0 10 stimulator output

0.5 0.5 percentage maximun 0 left right left right MEP amplitude (mV) MEP amplitude (mV) hemisphere 0.0 0.0 F task task task 1.6 Input-output curve no task no task no task 1.2 D Effect of time E Left hemisphere Right hemisphere 2.0 2.0 0.8 *** *** *** 0.4 1.5 1.5 MEP amplitude (mV) 0.0 1.0 1.0 90 100 110 120 130 140 stimulus intensity (% MT) 0.5 0.5 Fluent speakers MEP amplitude (mV) MEP amplitude (mV) Adults who stutter 0.0 0.0 All subjects first last first last first last Figure 5 Motor cortex excitability. (A and B) The tongue motor cortex is more excitable during resting state within the experimental speech trial (task) compared to resting state during the recording of the input-output curve (no task). Stimulation intensity was 120% of the individual motor threshold in both conditions. Depicted are MEP amplitudes averaged across all subjects. Grand average MEP amplitudes for fluent speakers and adults who stutter are comparable in magnitude within conditions and within hemispheres. (C) Motor thresholds are plotted as percentage of the maximum stimulator output. Adults who stutter exhibit higher motor thresholds than fluent speakers. (D and E) To ex- haustively test whether speaking with the mouthpiece affects excitability of the tongue motor cortex, we compared grand average amplitudes of Downloaded from the first 20 MEPs with the last 20 MEPs acquired in the transition phase after the second go-signal. The first MEP amplitudes were larger in magnitude compared to the last MEP amplitudes. When separated for hemisphere and group, only left hemisphere stimulation evoked larger MEP amplitudes in fluent speakers than in adults who stutter. (F) Shown are group mean MEP amplitudes of the input-output curve averaged over both

hemispheres. MEP amplitudes increase with rising stimulus intensity. The contralateral projection of the tongue (solid line) shows a slightly by guest on January 18, 2015 stronger attenuation compared to the ipsilateral projection (dashed line). Error bars indicate the standard error of mean. Asterisks indicate signiﬁcance levels: *P 5 0.05, ***P 5 0.001.

normalized, absolute MEP amplitudes taken from the tran- the two groups is the larger MEP response to left hemi- sition phase at the beginning (first 20 MEPs) and at the end spheric TMS in the control group (Fig. 5E). This is the of the test phase (last 20 MEP). ANOVA showed an effect effect that we also found in the normalized data and of Time [F(1,24) = 10.12, P=0.004, Greenhouse-Geisser report in Fig. 4B. corrected], an effect of Hemisphere [F(1,24) = 113.95, P 50.001], an effect of Group [F(1,24) = 26.0, P 5 0.001] and a Group Â Hemisphere interaction [F(1,24) = 26.18, P 5 0.001]. To further disentangle the Group Â The magnitude of left-lateralized Hemisphere interaction we calculated two mixed-model facilitation was correlated with ANOVAs separately for each hemisphere. Left hemisphere analysis yielded an effect of Time [F(1,24) = 6.31 P=0.019, stuttering severity Greenhouse-Geisser corrected] and an effect of Group If the observation of a modulated excitability of left hemi- [F(1,24) = 47.93, P 5 0.001] but no Group Â Time interac- sphere primary motor tongue representation was indeed tion. Right hemisphere analysis yielded an effect of Time related to speech fluency, one would expect a correlation [F(1,24) = 12.97 P=0.001, Greenhouse-Geisser corrected] between the excitability modulation and stuttered syllables. but no effect of Group and no interaction. Regardless of To assess this relationship, we plotted these parameters hemisphere or group, MEPs were smaller at the end of the against one other and observed a tight covariance, which experiment than at the beginning, as shown in Fig. 5D. In could be tentatively described by an exponential fit (Fig. 6). summary, the observed decrease in MEP amplitude from In this phenomenological description, the correlation be- the first third of test pulses in the transition phase to the tween the M1 excitability modulation and the percentage last third of pulses could indicate habituation. However, of stuttered syllables was significant for the left projection this occurs to the same degree in fluent speakers and (P=0.004) but only marginally significant for right projec- in adults who stutter. The finding that differentiates tions (P=0.055). 10 | BRAIN 2015: Page 10 of 14 N. E. Neef et al.

3.5 Fluent speakers 3.5 Adults who stutter 3.0 3.0 Left M1 projections 2.5 2.5 Right M1 projections 2.0 2.0 Exponential fits Right projections 1.5 1.5 Left projections 1.0 1.0 MEP amplitude 0.5 0.5 R² = 0.55 P = 0.004 R² = 0.30, P = 0.055 0.0 0.0 normalized to prefix baseline 0 1 2 3 0 5 10 15 20 25 stuttered syllables (%) stuttered syllables (%) Figure 6 Stuttering severity negatively correlated with speech induced facilitation. In adults who stutter, regression analysis yielded a signiﬁcant negative correlation between percentage of stuttered syllables in a speech sample of 1000 syllables and lack of facilitation of the MEP in the transition phase between speech gestures.

Adults who stutter have an input-output curve (Fig. 5F) yielded a main effect of Tongue projection [ANOVA, F(1,23) = 10.15, P=0.004], attenuated motor threshold of the a main effect of Intensity [F(5,19) = 33.1, P 5 0.0001], primary motor tongue and an interaction of Tongue projection Â Intensity representation [F(5,19) = 4.5, P=0.028], but no effect of Group [F(1,23) = 0.01, P=0.91] as well as no other effects. As The motor threshold was assessed to adjust stimulation in- shown in Fig. 5F, MEP peak-to-peak amplitudes increase tensity individually and is given as per cent of the maximum

with increasing stimulation intensity. In both groups, the Downloaded from stimulator output. This is a well-established procedure to facilitation was particularly marked in the contralateral avoid a group effect induced by different motor thresholds tongue projection after stimulation of the left hemisphere of the recruited subjects. The motor thresholds differed be- as shown in Fig. 5F. tween groups [ANOVA, effect of group, F(1, 24) = 6.09, The MEP input-output curve mirrors cortical excitability by guest on January 18, 2015 P 5 0.021], with no effect of hemisphere, and no interaction under resting state conditions. To investigate a potential (Fig. 5C). Post hoc t-tests demonstrated higher thresholds in effect due to speaking with the mouthpiece we compared adults who stutter (left 41.9, SD 7.3; right 42.6, SD 5.6) MEP amplitudes before and after familiarization. Before compared to ﬂuent speakers [left 37.6, SD 3.2; right 37.7, familiarization, as part of the input-output curve, MEPs SD 3.4; t(50) = À3.33, P=0.0016 with Bonferroni correc- were elicited at 120% motor threshold. The same intensity tion]. This difference should be interpreted carefully. was applied throughout the experimental phase and the Macroanatomical features dominate the motor threshold within-trial resting state condition, 2900 ms post-trial (Kozel et al., 2000; Salvador et al., 2011) and the intersub- onset. The mixed-model ANOVA resulted in an effect of ject variability is high, especially for stimulation of neurons Task [F(1,24) = 18.0, P 5 0.001] with an increased excit- of the primary motor tongue area, which extend into the ability observed in the within-trial condition (Fig. 5A and depth of the central sulcus’ ventrorostral part (Grabski B). No other effects or interactions occurred. This might be et al., 2012). Possibly due to the high intersubject variability, caused by a familiarization to the new speech environment previous reports on motor thresholds in stuttering are am- (Xivry et al., 2013; Kadota et al., 2014), or by training biguous (Neef et al.,2011a; Alm et al.,2013). Throughout (Svensson et al., 2006). An alternative cause could be a the experiment, the applied stimuli were normalized to the general arousal effect due to the visual attention required subjects motor threshold. This levels the intersubject variabil- in the task (Ruge et al., 2014). However, this occurred to ity and as a result, input-output curves show no effect of the same degree in ﬂuent speakers and in adults who stutter group, as reported in the next section. and was therefore not discussed.

In resting state, adults who stutter Discussion exhibit a normal dynamic range of motor cortex excitability Fluent speech requires the highly integrated control of the orofacial and respiratory muscles. During speech the oro- The MEP input-output curve was assessed to ensure a facial primary motor cortex integrates excitatory and in- broad dynamic range of excitability modulation in both hibitory signals from various cortical and subcortical sites groups and thus to avoid ceiling effects due to saturation in order to generate excitatory patterns orchestrating of the stimulated neural populations at a stimulation inten- speech movements. Speech flow is often interrupted in stut- sity of 120% of motor threshold. The analysis of the MEP tering, as sound prolongations, sound and syllable M1 tongue area excitation during speech BRAIN 2015: Page 11 of 14 | 11 repetitions, and speech blocks indicate an inability to move the primary motor cortex of fluent speakers waiting for the onto subsequent speech segments. Using TMS, we tracked initiation to release. This interpretation is supported by the modulation of motor cortex excitability during pro- the fact that phonological manipulation specifically involves cesses of speech motor planning and the timed initiation the left primary motor cortex but not the right primary of sensorimotor speech programs. Specifically, we acquired motor cortex in fluent speakers (Peeva et al., 2010). motor cortex excitability (i) during voluntary prolongation The fact that adults who stutter did not generate a facili- of a speech sound; and (ii) during the transition phase be- tation of left motor cortex is likely related to weaker struc- tween an invariant articulatory configuration and various tural connectivity and altered interplay between left subsequent speech movements. The major finding of our hemisphere speech-related brain regions. A huge body of current study was the unilateral facilitation of the orofacial literature supports the theory that stuttering is a disconnec- primary motor cortex in fluent speakers but the absence of tion syndrome because white matter integrity is reduced in such facilitation in adults who stutter. The critical patho- the fibre bundles of the left hemisphere speech-related net- physiological role of the primary motor cortices is stressed work (Sommer et al., 2002; Chang et al., 2008; Watkins by the inverse correlation of stuttering frequency and inter- et al., 2008; Cykowski et al., 2010; Cai et al., 2014). In a gestural facilitation (Fig. 6). fibre-tracking study (Chang et al., 2011), adults who stutter showed a significantly smaller number of white matter How can we explain the left voxels passing through the motor cortex in the left superior lateralized increase of motor cortex longitudinal fasciculus compared to fluent speakers. This structural deficit might impede the transfer of selected sen- excitability in fluent speakers and the sorimotor programs from left BA44/BA6 to the orofacial lack thereof in adults who stutter? motor cortex and, as a consequence, motor planning could exert a smaller influence on motor cortex excitability via The complex interactions of brain areas and the results of cortico-cortical connections (Greenlee et al., 2004). An al- related experiments are best discussed in the context of ternative view is that the reduced white matter integrity Downloaded from concrete models of cognitive function. The GODIVA hampers cortico-striatal connectivity (Civier et al., 2013) model (Gradient Order Directions into Velocities of interrupting the smooth sequencing of subsequent speech Articulators; Bohland et al., 2010) postulates two inde-

segments via the cortico-thalamo-cortical loop (Alm, 2004). by guest on January 18, 2015 pendent mechanisms in the primary motor cortex finalizing The lack of excitability facilitation found here in adults the cortical and subcortical control of fluent speech output: who stutter could also be caused by an imbalanced neuro- (i) motor output cells; and (ii) motor plan cells. Motor transmitter system because dopamine affects cortical excit- output cells coordinate the timing and initiation of sensorimotor speech programs. Here the pre-supplementary motor ability and activity in a non-linear, dose-dependent manner area triggers the release of planned speech sounds in inter- (Nitsche et al., 2010; Lissek et al., 2014). Indeed, in adults action with the putamen, globus pallidus, and thalamus. who stutter, increased dopaminergic activity has been re- This cortico-striato-thalamo-cortical network interacts in ported (Wu et al., 1997) and pharmacological interventions both the left and the right primary motor cortex to bilat- with dopamine antagonists have been shown to improve erally innervate the orofacial structures during speaking. In symptoms of stuttering (Maguire et al., 2004). When contrast to the motor output cells, the motor plan cells are given to control subjects, the dopamine agonists bromo- hypothesized to occur only in the left orofacial motor criptin and carbergoline reduce intracortical facilitation at cortex, which is supported by the observation of a left resting state (Ziemann et al., 1997; Korchounov et al., hemisphere specialization for phonation and vocalization 2007). In an earlier study we found that in adults who (Terumitsu et al., 2006). The motor plan cells integrate stutter the intracortical facilitation is likewise reduced selected sensorimotor programs driven by the left posterior (Neef et al., 2011b). This complementary neurobiological inferior frontal gyrus, the adjoining left ventral premotor theory of stuttering supports the view that a chronic hyper- cortex [Brodmann area (BA) 44/BA6], and the left supple- dopaminergic activity could hinder response competition mentary motor area (Peeva et al., 2010). An uncoupling of and thus the timed selection of motor output cells (Civier motor output cells from motor plan cells would allow the et al., 2013) and also connects to the findings of impaired system to prepare subsequent sensorimotor programs while motor learning in adults who stutter (Smits-Bandstra et al., a former program is executed (Bohland et al., 2010). 2006; Smits-Bandstra and De Nil, 2009). Our data show that the excitability of the left orofacial In either case, disconnections, elevated dopamine or a motor cortex is transiently increased just before a novel combination of both might result in a weakened transfer speech gesture is executed. It thereby provides evidence of sensorimotor programs to the motor plan cells in the left for such an uncoupling of speech motor planning in the M1 or effect a delayed selection of motor output cells. The left primary motor cortex from speech output initiation in reduced dynamic range for intracortical facilitation indi- both motor cortices. It is tempting to speculate that the cated a weakening of the capability to encode specialized increased excitation of the left orofacial motor cortex in sensorimotor programs at the level of the primary motor the transition phase mirrors activated motor programs in cortex. Both processes seem to be plausible mechanisms 12 | BRAIN 2015: Page 12 of 14 N. E. Neef et al. that could result in intermittent interruptions of fluent 0.19 mV; t(24) = 6.629, P 5 0.001]. Right hemisphere MEP speech. amplitudes were not different between groups (fluent speakers 1.06 mV, SD 0.17 mV; adults who stutter 1.0 mV, SD 0.18 mV). The dramatic effect is in line with our interpret- Speech sound prolongation elevates ation and as it occurs in the raw MEP amplitudes, it ex- left orofacial primary motor cortex in cludes the possibility that the normalization created the stuttering effect of group. However, for the sake of our study question we decided The voluntary prolongation of a speech sound is a rather to normalize MEP amplitudes of the transition phase to the artificial condition for fluent speakers, whereas it happens state of cortical excitability during prolongation. MEPs are involuntarily in stuttering. In the current experiment, we variable in size and shape even when stimulation intensity asked adults who stutter as well as fluent speakers to vol- and the coil positioning are kept constant (Ro¨ sler et al., untarily prolong the fricative of the German prefix ‘auf’ in 2008). This variability is particularly pronounced when re- order to acquire the state-dependent motor cortex excitabil- cording from orofacial muscles (Devlin and Watkins, ity for speaking. Accordingly, we controlled for cognitive 2007). The neurogenic origin of this variability lies partly load and pre-TMS innervation of orofacial muscles. The in the fluctuating number of excited motor neurons as a articulation of the fricative requires a constriction between result of variation of facilitation by voluntary contraction the lower lip and the upper incisors, but the tongue rests or cognitive events. Speaking integrates both voluntary con- with its tip gently touching the lower incisors. Although the traction of orofacial muscles and cognitive control. During tongue does not play an active part in this, motor cortex prolongation of the introductory syllable ‘auf’ the orofacial excitability was increased compared to the resting state cortical neurons descend into a reference state of excitabil- (Fig. 3). Considering the bilateral innervation of articula- ity, an appropriate reference to normalize MEP amplitudes tory structures, one would expect a comparable increase of during the transition phase. excitability in both hemispheres. But our data show a left- Downloaded from lateralized increase in adults who stutter, contrary to a trend towards a right lateralized increase in fluent speakers. Conclusion and outlook Besides the prolongation of the prefix ‘auf’, the cognitive Our data integrate structural and neurophysiological find- state during this time in the task also reflects preparation ings into a plausible model of speech pathophysiology in by guest on January 18, 2015 for the production of the next word following the second persistent developmental stuttering. Moreover, our results go signal. Therefore the observed elevated excitation may support a theoretical framework of speech motor control, reflect a different preparatory behaviour with an excessive namely the GODIVA model (Bohland et al., 2010). With activation of the primary motor cortex instead of the infer- regard to this model, our data suggest that stuttering is ior frontal areas in adults who stutter (Salmelin et al., associated with the inadequate activation of sensorimotor 2000). plans in the left orofacial motor cortex. Because it is pos- This observed group difference during the state we nor- tulated that cortico-cortical connections from BA44/BA6 malized to may raise the question whether normalizing the and the supplementary motor area provide the segmental data of the transition phase to the phase of prolongation and suprasegmental plan, one could speculate that a facili- could have driven the apparent differences in MEPs during tation of those regions, for instance with non-invasive neu- speech production. To demonstrate that between-group dif- rostimulation, would facilitate the stable activation of the ferences were not driven by the normalization, we plotted sensorimotor plans in the left primary motor cortex. This the grand averages of the non-normalized absolute MEP should be evident in a normalization of the motor cortex amplitudes for all conditions in Fig. 4A. Kolmogorov- excitability in the transition phase between articulatory Smirnov tests indicated non-normality of MEP amplitudes gestures. in the bins characterizing the response-locked trajectory of Stuttering was not overtly present in the vast majority of speech motor preparation. To overcome this problem of our trials. Our finding therefore indicates an abnormal variability, we pooled absolute MEP amplitudes for all motor preparation in adults who stutter, even in mostly time points before speaking but separately for each hemi- fluent parts of speech. This points to a chronic aberration sphere. The same data points that entered the analysis on of the physiology not permanently accompanied by clinical normalized data were analysed here. An additional mixed- symptoms (Fox et al., 1996). Our method allows probing model ANOVA with Hemisphere as within-subjects factor the functional integrity of speech production networks in a and Group as between-subjects factor resulted in an effect performance independent manner. of Group [F(24,1) = 18.86, P 5 0.001], an effect of Our data do not allow conclusions with regard to sub- Hemisphere [F(24,1) = 140.24, P 5 0.001, Greenhouse- cortical pathological mechanism of stuttering. This is prob- Geisser corrected], and a Group ÂHemisphere interaction lematic because a large body of literature emphasizes [F(24,1) = 70.22, P 5 0.001]. Left hemisphere MEP ampli- subcortical irregularities (Wu et al., 1997; Alm, 2004; tudes were significantly larger in fluent speakers (1.67 mV, Brown et al., 2005; Giraud et al., 2008; Chang et al., SD 0.25 mV) compared to adults who stutter [1.12 mV, SD 2011; Ingham et al., 2012). A combination of a virtual M1 tongue area excitation during speech BRAIN 2015: Page 13 of 14 | 13 lesion approach with our new method would allow one to Brown S, Ingham RJ, Ingham JC, Laird AR, Fox PT. Stuttered and indirectly prove the impact of the timed initiation of speech fluent speech production: an ALE meta-analysis of functional neuroimaging studies. Hum Brain Mapp 2005; 25: 105–17. gestures and thus the functionality of the cortico-striato- Busan P, D’Ausilio A, Borelli M, Monti F, Pelamatti G, Pizzolato G, thalamo-cortical loop. Inhibition of the pre-supplementary et al. Motor excitability evaluation in developmental stutter- motor area as part of this loop would hinder the balance of ing: a transcranial magnetic stimulation study. Cortex 2013; 49: input from the thalamus to the bilaterally organized motor 781–92. output cells. If this balance played a role in the planning Cai S, Tourville JA, Beal DS, Perkell JS, Guenther FH, Ghosh SS. phase, one would observe a decrease of facilitation in the Diffusion imaging of cerebral white matter in persons who stutter: evidence for network-level anomalies. Front Hum Neurosci 2014; 8: left primary motor cortex of fluent speakers. Likewise, a 54. facilitation of this loop would normalize the left motor Chang SE, Erickson KI, Ambrose NG, Hasegawa-Johnson MA, cortex excitability in adults who stutter. It will be import- Ludlow CL. Brain anatomy differences in childhood stuttering. ant to test these hypotheses in future experiments. Neuroimage 2008; 39: 1333. Chang S-E, Horwitz B, Ostuni J, Reynolds R, Ludlow CL. Evidence of Left Inferior frontal–premotor structural and functional connectivity deficits in adults who stutter. Cereb Cortex 2011; 21: 2507–18. Acknowledgements Chang S-E, Zhu DC. Neural network connectivity differences in children who stutter. 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Mechanisms of human motor cortex facilitation induced by subthreshold 5-Hz repetitive transcranial magnetic stimulation

Martin Sommer,1 Milena Rummel,1 Christoph Norden,1 Holger Rothkegel,1,2 Nicolas Lang,1,2 and Walter Paulus1 1Department of Clinical Neurophysiology, University of Goettingen, Goettingen, Germany; and 2Department of Neurology, University of Kiel, Kiel, Germany

Submitted 19 December 2012; accepted in ﬁnal form 27 March 2013 Downloaded from

Sommer M, Rummel M, Norden C, Rothkegel H, Lang N, ties below the motor threshold ensures a mostly cortical site of Paulus W. Mechanisms of human motor cortex facilitation induced action (Bestmann et al. 2004; Huang et al. 2005), particularly by subthreshold 5-Hz repetitive transcranial magnetic stimulation. J when the intensity is below the active motor threshold (AMT), Neurophysiol 109: 3060–3066, 2013. First published March 27, 2013; i.e., too weak to activate the corticospinal tract even during http://jn.physiology.org/ doi:10.1152/jn.01089.2012.—Our knowledge about the mechanisms voluntary activation of the target muscle (Quartarone et al. of human motor cortex facilitation induced by repetitive transcranial magnetic stimulation (rTMS) is still incomplete. Here we used phar- 2005; Sommer et al. 2012). macological conditioning with carbamazepine, dextrometorphan, We have further characterized this facilitation outlasting lorazepam, and placebo to elucidate the type of plasticity underlying subthreshold rTMS pulses by showing that they are best this facilitation, and to probe if mechanisms reminiscent of long-term induced by using biphasic pulses inducing effective posteriorly potentiation are involved. Over the primary motor cortex of 10 healthy directed currents in the motor cortex (Rothkegel et al. 2010; subjects, we applied biphasic rTMS pulses of effective posterior Sommer et al. 2012). However, this specific facilitation elicited current direction in the brain. We used six blocks of 200 pulses at even with such low intensity was of rather short duration, only 5-Hz frequency and 90% active motor threshold intensity and con- some 15 min, shorter than has been reported with stronger at Nds. Staats-und Universitaetsbibliothek on June 17, 2013 trolled for corticospinal excitability changes using motor-evoked stimuli (Pascual-Leone et al. 1994) or theta burst stimulation potential (MEP) amplitudes and latencies elicited by suprathreshold (Huang et al. 2005). We were skeptical as to whether mecha- pulses before, in between, and after rTMS. Target muscle was the dominant abductor digiti minimi muscle; we coregistered the domi- nisms reminiscent of long-term potentiation (LTP) would be nant extensor carpi radialis muscle. We found a lasting facilitation involved in that type of plasticity. induced by this type of rTMS. The GABAergic medication lorazepam We therefore tried to further characterize the nature of and to a lesser extent the ion channel blocker carbamazepine reduced this facilitation by testing its sensitivity to pharmacological the MEP facilitation after biphasic effective posteriorly oriented conditioning. In particular, we evaluated N-methyl-D-aspar- rTMS, whereas the N-methyl-D-aspartate receptor-antagonist dex- tate (NMDA) antagonism, known to reduce the induction of trometorphan had no effect. Our main conclusion is that the mecha- LTP (Netzer et al. 1993), carbamazepine (CBZ), known to nism of the facilitation induced by biphasic effective posterior rTMS block voltage-dependent sodium channels (White 1997), is more likely posttetanic potentiation than long-term potentiation. and lorazepam (LOR), known to block many neuroplastic Additional findings were prolonged MEP latency under carbamaz- processes (Treiman 2001; White 1997). epine, consistent with sodium channel blockade, and larger MEP amplitudes from extensor carpi radialis under lorazepam, suggesting GABAergic involvement in the center-surround balance of excitabil- METHODS ity. Participants and drug conditions. The study protocol was approved transcranial magnetic stimulation; plasticity; dextrometorphan; carba- by the University of Goettingen ethics committee, and written, in- mazepine; lorazepam formed consent was obtained from all participants. We conducted a five-armed double-blind placebo (PLC)-controlled pharmacological study in 10 healthy, right-handed subjects (six women, mean age 23.9, REPETITIVE TRANSCRANIAL MAGNETIC stimulation (rTMS) modu- range 19–35 yr; mean Oldfield handedness score of 91.5, standard lates corticospinal excitability and is widely used in many deviation 21.5 points; no regular intake of central nervous system- disciplines, including clinical trials (Janicak et al. 2010; active medication). Subjects were recruited from the University cam- O’Reardon et al. 2007) and FDA approval in depression pus and paid for participation. They were free to withdraw participation at any point. They were instructed on the blinded testing of (Melkerson 2008). Though the motor cortex has been abun- different, well-established centrally active drugs and PLC, and they dantly studied using rTMS (Pell et al. 2011; Ridding and were informed about common side effects and asked to report even- Ziemann 2010), our knowledge about the mechanisms of tual side effects. The sample size was based on the presence or action of rTMS applied in humans is still limited. In particular, absence of an rTMS effect with regard to the first five bins after the studies elucidating mechanisms of rTMS by pharmacological two types of biphasic rTMS shown in Fig. 1 of Sommer et al. (2012), conditioning are scarce. yielding an ␩2 of 0.19 and an effect size of f ϭ 0.48, with a correlation It is long known that rTMS can induce a lasting facilitation between repeated measures of 0.6. A power analysis (Gpower 3.1.5, of the corticospinal tract, and that the use of stimulus intensi- University of Kiel, Germany) for repeated-measures ANOVA based on four measurements in one group indicated that with nine subjects Address for reprint requests and other correspondence: M. Sommer, Dept. of our study would have a power of 95%. The same power can be Clinical Neurophysiology, Univ. of Goettingen, Robert-Koch-Str., 40, D-37075 achieved in a pre-post comparison at the usual two-sided significance Goettingen, Germany (e-mail: [email protected]). level of 5%, with a same sample size of nine subjects, as long as the

3060 0022-3077/13 Copyright © 2013 the American Physiological Society www.jn.org POSTTETANIC POTENTIATION IN THE HUMAN MOTOR CORTEX 3061 standardized effect size is at least 1.4, which is supported by findings configuration because of a technical defect in the new MagPro of Quararone and colleagues (2005) on 5-Hz rTMS in healthy sub- stimulator that we specified elsewhere (Sommer et al. 2006; see jects. In our study, the participants were studied in the morning 12 h editorial Valls-Solé and Hallett 2006). after an evening oral dose of medication and 2 h after a morning oral Transcranial magnetic stimulation procedure. During the experi- dose of medication. The CBZ condition consisted of 300 mg of CBZ mental sessions, participants were sitting comfortably in a reclining in the evening and in the morning, the LOR condition of 2 mg LOR chair with the arms and neck supported. EMG recordings of the in the morning preceded by PLC in the evening, the dextrometorphan abductor digiti minimi muscle (ADM) and the extensor carpi radialis (DMO) condition of 150 mg DMO in the morning preceded by PLC (ECR) muscle on the side of the dominant hand were recorded by in the evening, and the PLC condition of PLC both in the evening and separate pairs of silver-silver chloride electrodes in a belly-tendon in the morning. There was at least 1-wk delay in between measure- montage. The ADM served as target muscle; the more proximal ments to account for possible carry-over effects, and the order of muscle was studied to assess a possible dissociation between excit-

experiments was randomized. Serum levels (Geradin et al. 1976; ability changes in the cortical representation of proximal and distal Downloaded from Greenblatt et al. 1979; Silvasti et al. 1987) and central nervous effects muscles (Tings et al. 2005). For recording we used the “Signal” of these drugs at these dosages can be expected to peak about 2 h after software at a sampling rate of 10 kHz, and filtered at 1.6 Hz and 1 kHz oral intake (Ziemann et al. 1996a, 1996b, 1998a). CBZ is better (CED 1401, Cambridge Electronic Design, Cambridge, UK). To tolerated if taken in two consecutive dosages (Nitsche et al. 2003). control for muscle relaxation, audiovisual feedback was provided, and To elucidate the mechanisms of the facilitation observed after 5-Hz 50 ms of pre-TMS EMG were recorded during each trial. The coil rTMS, we used biphasic pulses with effective current direction in the was hand held over the dominant hemisphere by the investigators brain from anterior to posterior (biphasic p-a/A-P, named “biphasic-P”) (M. Rummel or M. Sommer). The coil position was frequently http://jn.physiology.org/ in four conditions (LOR, CBZ, DMO, PLC). We highlight the second controlled and corrected, if necessary. Hence, participants were under component of the induced current (Sommer et al. 2012) covering the constant supervision, which allowed ensuring a stable level of atten- second and third quarter cycles (Kammer et al. 2001; Sommer et al. tion throughout the experiment. 2006), which is more important than the initial quarter cycle Biphasic and half-sine TMS pulses were generated by a MagPro (Maccabee et al. 1998; Salvador et al. 2011), primarily because of its X100 MagOption (Medtronic, Minneapolis, MN) stimulator con- longer duration (Maccabee et al. 1998). For the sake of clarity, the nected to a slightly bent figure-of-eight coil (MC B70, Dantec S.A., current directions indicated in this paper always refer to the induced Skovlunde, Denmark). In each session, the suprathreshold stimuli current flow in the brain. delivered before and after rTMS and in between rTMS blocks were of CBZ blocks voltage-dependent sodium channels and high-fre- the same type and orientation as the rTMS pulses. We determined the quency spiking (White 1997). In the human corticospinal tract, it optimal dominant hand motor representation of the ADM by applying at Nds. Staats-und Universitaetsbibliothek on June 17, 2013 raises the motor threshold, but does not have a pronounced effect on single TMS pulses with the coil held perpendicular to the presumed intracortical inhibition or facilitation (Ziemann et al. 1996b). CBZ localization of the central sulcus and with the handle pointing back- blocks rTMS-induced motor-evoked potential (MEP) facilitation (In- wards at about 45° laterally. The position yielding the highest MEP ghilleri et al. 2004). LOR is a benzodiazepine reinforcing GABAA amplitudes was marked with a pen. The resting motor threshold action on chloride channels by modulating the channel opening (RMT) with regard to the ADM was determined by delivering single frequency (Treiman 2001; White 1997). It enhances short latency TMS pulses over the marked position and by reducing the stimulus intracortical inhibition and reduces intracortical facilitation, but leaves intensity in steps of ϳ1% stimulator output. RMT was defined as the the motor threshold mostly unchanged (Ziemann et al. 1996a). It lowest intensity at which at least 5 out of 10 consecutive MEPs were reduces plasticity in experimental (Fitzgerald et al. 2005; Werhahn Ͼ50 ␮V in amplitude with the subject at rest. Similarly, we deter- et al. 2002; Ziemann et al. 2001) and clinical settings (Conca et al. mined the AMT as the minimal intensity evoking MEP amplitudes in 2000; Teo et al. 2009; Wan et al. 2004), e.g., after stroke, thereby the ADM of at least 250 ␮V in 5 of 10 consecutive trials during slight worsening the functional recovery of the patients (Lazar et al. 2003). voluntary contraction of the target muscle (Rossi et al. 2009; Rothwell DMO is an NMDA receptor antagonist, and at high dose levels it also et al. 1999). has channel-blocking properties (Netzer et al. 1993). It reduces intra- To determine a baseline of corticospinal excitability, rTMS was cortical facilitation and has no major effect on the motor threshold preceded by four consecutive bins of 30 single pulses at 0.25 Hz at an (Ziemann et al. 1998a). DMO blocked plasticity in a variety of intensity adjusted to yield baseline MEP amplitudes in the ADM of experimental settings (Fitzgerald et al. 2005; Stefan et al. 2002; about 1 mV. Immediately following this baseline, six blocks of 200 Wolters et al. 2003). pulses each were delivered at 5-Hz frequency (Sommer et al. 2012). All drugs were prepared by the pharmacy of the University of The intensity of 5-Hz stimulation was 90% AMT of the ADM. During Goettingen, packed in identical blue capsules and delivered in sepa- each interval of 2 min in between the rTMS blocks, 15 pulses of rate, clearly designated jars. Medication for each experiment of each baseline intensity were applied at 0.25-Hz frequency. After the end of subject was packed in a blister containing the evening and the rTMS, this assessment of corticospinal excitability was prolonged for morning dose in separate spaces by a physician not involved in data 12 consecutive bins of 30 pulses at 0.25 Hz and of baseline intensity recording and analysis (N. Lang), and each blister was marked with to detect possible lasting effects. Figure 1 illustrates this experimental the subject number and experiment number. The subjects and the design. We used subthreshold rTMS pulses in these experiments investigators involved in data recording (M. Rummel and M. Som- because 1) earlier trials with suprathreshold pulses at 5 Hz were mer) were blinded to the drug status during data acquisition and limited in duration of stimulation by safety concerns (Tings et al. analysis. Only after off-line measurement and normalization and 2005); and 2) we intended to ensure a mostly cortical site of effect (Di averaging of motor threshold, MEP amplitudes, and latencies was Lazzaro et al. 2004). All these procedures were done anew for each blinding broken to allow calculating statistics. We did not control for session, to account for eventual drug effects on threshold eventually serum levels, which is a limitation of this study, but asked each requiring adjustments of stimulus intensity. subject at each experiment for a detailed report of side effects, which Data analysis. For analysis, we normalized the individual mean were meticulously noted. amplitudes of each bin to the corresponding baseline. In a first step, To further confine the specificity of the observed facilitation we compared the two PLC conditions using a repeated-measures (Sommer et al. 2012) in a distinct group of subjects, we used half-sine ANOVA across all time bins with muscle and time as well as well as initially posteriorly oriented pulses [half-sine a-p (Sommer et al. type of rTMS as within-subjects factors. In a second step, we com- 2006)] in one additional PLC condition. We intended to use mono- pared the four conditions for biphasic effective posteriorly oriented phasic pulses for comparison, these turned out to be of half-sine pulses (CBZ, LOR, DMO, PLC) using a repeated-measures ANOVA

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Evening 20:00 hrs Morning 08:00 hrs 10:00 hrs

b1 ... b4 i1 ... i5 in between p1.... p12 2 ... 8 min 5 Hz rTMS 2 .... 24 min 6 blocks à 200 pulses

carbamazepine carbamazepine 52.0 zH biphasic P sa enilesab

placebo dextrometorphan biphasic P

placebo lorazepam biphasic P

placebo placebo biphasic P Downloaded from

placebo placebo half sine a-p

Fig. 1. Schematic diagram of study protocol. rTMS, repetitive transcranial magnetic stimulation; P, posterior; a-p, anterior to posterior. with muscle and time as well as drug as within-subject factors. Here, dizziness (2), mnestic difficulties (2), nausea (2); CBZ: dizzi- effects during and after rTMS were analyzed separately, focusing on ness (7), fatigue (5), nausea (2), ataxia (1); DMO: ataxia (7), the last two bins during rTMS and the first two bins after rTMS where dizziness (7), nausea (7), euphoria (4), fatigue (1). These side http://jn.physiology.org/ a maximum rTMS effect could be expected (Sommer et al. 2012). Based on significant main effects, we compared each bin of MEPs effects reached their maximum within 1–4 h after intake and during rTMS to baseline with exploratory two-tailed, paired t-tests. were fully reversible until the morning after the experiment. Drug effects on the baseline MEP amplitudes were assessed on None of the subjects opted to drop out of the study. individual mean baseline MEP amplitude using a repeated-measures Motor threshold, MEP amplitude, and MEP latency. The ANOVA with drug and muscle as between-subjects factors. Based on motor threshold was higher after CBZ intake than at any other significant main effects or interactions, exploratory post hoc t-tests drug condition [repeated-measures ANOVA, effect of drug, compared drug effects, separate for each muscle. F(3,27) ϭ 3.61, P ϭ 0.026, ␩2 ϭ 0.02; effect of voluntary Drug effects on the baseline RMT and AMT as well as the mean ϭ Ͻ ␩2 ϭ at Nds. Staats-und Universitaetsbibliothek on June 17, 2013 MEP amplitude were assessed using a repeated-measures ANOVA muscle contraction, F(1,9) 40.30, P 0.0001, 0.21; with drug and presence of voluntary muscle contraction (AMT, RMT) interaction of drug by innervation, F(3,27) ϭ 4.52, P ϭ 0.011, 2 as between-subjects factors. Based on significant main effects, post ␩ ϭ 0.005; Fig. 2]. Post hoc t-tests showed that the CBZ hoc, Bonferroni-corrected t-tests compared drug effects. We went on to condition yielded higher thresholds than the DMO condition. calculate similar ANOVAs separately for AMT and RMT. We measured The difference between CBZ and PLC slightly failed the the MEP onset latency for the ADM from the 21st to the 30th MEP of Bonferroni-correction threshold of the post hoc t-test. The the second baseline bin, chosen to ensure baseline stability (Tegent- relative increase between AMT and RMT was largest for CBZ hoff et al. 2005) and to minimize any putative influence of pre-TMS activity (Pasley et al. 2009), and the first bin after rTMS, and analyzed and smallest for DMO, but post hoc t-tests did not yield drug and rTMS effects on the MEP latency using a repeated-measures significance. ANOVA with drug and time as within-subjects factors. With regard to Separate ANOVAs for RMT and AMT confirmed a drug half-sine pulses, AMT, RMT, and latency are reported in Fig. 2, but effect on RMT [repeated-measures ANOVA, effect of drug, not included in the analysis. For all significant main effects or F(3,27) ϭ 6.03, P ϭ 0.04, ␩2 ϭ 0.04], but not on AMT interactions of all analyses, we indicated the effect size ␩2 as calcu- [repeated-measures ANOVA, effect of drug, F(3,27) ϭ 1.09, lated from the SPSS 20.0 repeated-measures general linear model P ϭ 0.37]. (International Business Machines, Armonk, NY). Similarly, the stimulus intensity necessary to induce MEP amplitudes of about 1 mV was highest in the CBZ condition ϭ RESULTS [repeated-measures ANOVA, effect of drug, F(3,27) 4.28, P ϭ 0.014, ␩2 ϭ 0.04; Fig. 2]. Post hoc t-tests confirmed a Adverse events. Side effects reported by the subjects in- significant difference between CBZ and PLC, as well as cluded the following: LOR: fatigue (8 subjects), ataxia (5), between CBZ and DMO.

100 23.0

Fig. 2. Motor threshold, stimulus intensity, and 90 22.5 latency. Active motor threshold (AMT), resting motor threshold (RMT), stimulus intensity re- 80 quired to yield motor-evoked potential (MEP) AMT 22.0 Latency pre ϳ amplitudes of 1 mV, as well as MEP latency RMT 70 Latency post before and after rTMS plotted for each of the 1 mV 21.5 pharmacological conditions tested are shown. All [ms] 60 measures are taken from abductor digiti minimi [A/µs] (ADM) muscle recordings. Values are means Ϯ 21.0 SE. In addition, results of the half-sine pulses 50 control condition are shown. CBZ, carbamaz- 20.5 epine; DMO, dextromethorphan; LOR, loraz- 40 epam; PLC, placebo; hs, half-sine. 30 20.0 CBZ DMO LOR PLC PLC hs

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absence of facilitation with half-sine pulses under PLC corrob- 1.4 CBZ DMO LOR PLC orates the orientation specificity of this facilitation discussed 1.2 elsewhere (Sommer et al. 2012), i.e., that this facilitation is specifically induced with biphasic pulses that have a posterior 1.0 current flow in the brain with regard to their decisive second and third quarter cycle component (Maccabee et al. 1998). 0.8 Drug effects during rTMS. For the last two bins during 0.6 rTMS, no drug influence was found [repeated-measures ANOVA, effect of drug, F(3,27) ϭ 0.51, P ϭ 0.67; effect of 0.4 muscle, F(1,9) ϭ 0.85, P ϭ 0.38, no effect of time, no

0.2 significant interaction; Fig. 5]. Downloaded from Drug effects after rTMS. LOR, and to a lesser extent CBZ, 0 Raw baseline MEP amplitude [mV] diminished the MEP facilitation after biphasic effective poste- ADM ECR riorly oriented rTMS, whereas DMO had no major effect [repeated-measures ANOVA, effect of drug, F(3,27) ϭ 3.7, Fig. 3. MEP amplitudes at baseline. Average raw MEP amplitudes before ϭ ␩2 ϭ ϭ ϭ rTMS in the ADM and the extensor carpi radialis (ECR) muscles, for the four P 0.023, 0.12; effect of muscle, F(1,9) 6.4, P 2 drug conditions specified on the abscissa are shown. Values are means Ϯ SE. 0.033, ␩ ϭ 0.04, no effect of bin, no significant interaction; Fig. 5]. Post hoc t-tests yielded a significant difference between http://jn.physiology.org/ The MEP latency from ADM was longer under CBZ than LOR and PLC, as well as between LOR and DMO (P Ͻ 0.02); under any other condition; it was not altered by rTMS [repeat- ϭ ϭ the difference between CBZ and PLC slightly missed signifi- ed-measures ANOVA, effect of drug, F(3,27) 5.12, P cance (P ϭ 0.065). 0.006, ␩2 ϭ 0.17; effect of time, F(1,9) ϭ 0.01, P ϭ 0.92; no significant interaction; Fig. 2]. Post hoc t-tests confirmed a significant difference between CBZ and LOR, as well as DISCUSSION between CBZ and PLC. The facilitation observed for about 20 min after subthreshold The baseline MEP amplitude of ADM and ECR muscles are rTMS is blocked by LOR and reduced by CBZ, but not by shown in Fig. 3; they were not significantly different between DMO. There was no prolongation of the after-effects induced at Nds. Staats-und Universitaetsbibliothek on June 17, 2013 drug conditions in either muscle [repeated-measures ANOVA, by any of the drugs tested. effect of drug, F(3,27) ϭ 1.04, P ϭ 0.39; effect of muscle, CBZ also reduced the MEP facilitation induced by suprath- F(1,9) ϭ 0.003, P ϭ 0.99; interaction of muscle by drug, reshold 5-Hz rTMS (Inghilleri et al. 2004), which was attrib- F(3,27) ϭ 3.36, P ϭ 0.033, ␩2 ϭ 0.04, no other significant uted to its sodium-channel blocking properties. The blockage interactions; Fig. 3]. Post hoc t-tests separate for each muscle of the facilitation by CBZ is reminiscent of its effects on the yielded a significant difference between LOR and PLC for the facilitation of MEP amplitudes evoked by anodal transcranial ECR muscle only. direct current stimulation (tDCS) both during and after stimu- Comparison of PLC conditions. The analysis of the two PLC lation (Liebetanz et al. 2002; Nitsche et al. 2003). Again, this arms confirmed a facilitation with biphasic effective posteri- was explained by a blockage of voltage-dependent sodium orly oriented pulses, but not with half-sine initially posteriorly channels (Nitsche et al. 2003). Similarly, models of epilepsy oriented pulses [effect of rTMS type, F(1,9) ϭ 6.1, P ϭ 0.036, have shown a decreased spike frequency after kindling follow- ␩2 ϭ 0.07; effect of time, F(20,180) ϭ 2.1, P ϭ 0.0051, ␩2 ϭ ing treatment with CBZ (White 1997). Prolongation of evoked 0.05; no effect of muscle; interaction of type by time, potential latency by CBZ has been shown for laser evoked F(20,180) ϭ 2.44, P ϭ 0.001, ␩2 ϭ 0.05; see Fig. 4]. The potentials (Cruccu et al. 2001), but not for MEPs, to the best of

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1.0 Fig. 4. MEP amplitudes of PLC conditions. Shown are MEP amplitudes before, in between, and after six blocks 0.8 of rTMS; two types of PLC rTMS were studied in 10 subjects. Results from ADM are shown. Values are Ϯ Ͻ 0.6 means SE. *P 0.05 to baseline.

0.4 Biphasic P MEP amplitude normalised to baseline MEP amplitude normalised to 0.2 Half sine a-p

0 1234123 4 5 2 4 6 8 10 12 14 16 18 20 22 24 Bins at Bins between Minutes after rTMS baseline rTMS blocks

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0 http://jn.physiology.org/ Fig. 5. Pharmacological modulation of MEP amplitudes 1234123 4 5 2 4 6 8 10 12 14 16 18 20 22 24 changes. Shown are MEP amplitudes before, in between, Bins at Bins between Minutes after rTMS and after six blocks of rTMS; one type of rTMS was baseline rTMS blocks studied under preconditioning with DMO, CBZ, LOR, or PLC in 10 subjects. Results from ADM (A) and ECR B - extensor carpi radialis PLC CBZ DMO LOR muscle (B) are shown. Values are normalized to the individual mean of the baseline bins of each condition. 2.0 Values are means Ϯ SE. 1.8 at Nds. Staats-und Universitaetsbibliothek on June 17, 2013 1.6

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0 1234123 4 5 2 4 6 8 10 12 14 16 18 20 22 24 Bins at Bins between Minutes after rTMS baseline rTMS blocks our knowledge. The prolonged MEP latency under CBZ influ- et al. 2003), but not after shorter stimulation (Nitsche et al. ence may be related to a longer delay until a sufficient propor- 2003). For INB, it did not block the MEP facilitation, but the tion of motoneurons have reached their firing threshold (Roth- decrease of intracortical inhibition induced by INB in combi- well et al. 1991). nation with slow-frequency rTMS (Ziemann et al. 1998b). LOR clearly suppressed the short-lasting MEP facilitation. Taken together, the pharmacological profile observed in our When investigated in the context of tDCS, LOR delayed the study does not favor LTP as the underlying mechanism, since facilitation induced by anodal tDCS (Nitsche et al. 2004). It it depends on glutamate-mediated synaptic enhancement and reduced the facilitation of MEP amplitudes in conjunction with can be blocked in vitro by glutamate receptor antagonists a reduction of intracortical inhibition induced by the combined (Kirkwood et al. 1993), whereas DMO did not block the intervention of ischemic nerve block (INB) at the level of the facilitation observed here. An insufficient DMO dosage is elbow and slow rTMS over the corresponding motor cortex unlikely, since it was chosen according to the published liter- (Ziemann et al. 1998b). This and the appearance of latent ature (Liebetanz et al. 2002; Nitsche et al. 2003; Ziemann et al. cortico-cortical connections after GABA blockade in rats (Ja- 1998a) and elicited clinically relevant side effects. Short-term cobs and Donoghue 1991) suggest that the inhibitory activity potentiation (STP) probably has different underlying mecha- of GABA blocks many neuroplastic processes. nisms (Citri and Malenka 2008) and may lower the threshold DMO, in our study, did not modify the facilitation of MEP for LTP induction. However, one report claims that it is amplitudes. However, DMO abolished the lasting facilitation NMDA dependent as well (Schulz and Fitzgibbons 1997). after longer tDCS stimulation (Liebetanz et al. 2002; Nitsche Shorter lasting modulations include posttetanic potentiation

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(PTP), which is NMDA-receptor independent (Schulz and GRANTS Fitzgibbons 1997; Swandulla et al. 1991) and is diminished by This work was supported by the Deutsche Forschungsgemeinschaft Grants LOR (Brown et al. 1983) and by CBZ (Talwar 1990). Hence, SO429/2-1 and SO429/2-2 (M. Sommer), by the Bernstein Center for Com- STP and particularly PTP are a more likely candidate mecha- putational Neuroscience Grant 01GQ0432 (W. Paulus) and the Rose Founda- tion (W. Paulus). nism for the facilitation observed here. PTP has shown to depend essentially on presynaptic mechanisms such as an increased frequency of spontaneous neurotransmitter vesicle DISCLOSURES release (Habets and Borst 2005) and increase of calcium influx No conflicts of interest, financial or otherwise, are declared by the author(s). per action potential (Habets and Borst 2006). To our knowledge, a possible association of rTMS and PTP has not been AUTHOR CONTRIBUTIONS Downloaded from systematically studied before and has only been alluded to in Author contributions: M.S., C.N., H.R., N.L., and W.P. conception and the context of postexercise facilitation (Samii et al. 1996, design of research; M.S., M.R., and N.L. performed experiments; M.S., M.R., 1998). Inghilleri and coworkers (2004) implicated postsynaptic and C.N. analyzed data; M.S., N.L., and W.P. interpreted results of experiments; M.S. prepared figures; M.S. drafted manuscript; M.S., M.R., C.N., NMDA-receptor dependent mechanisms in their findings on a H.R., N.L., and W.P. edited and revised manuscript; M.S., N.L., and W.P. blockade of rTMS-related MEP facilitation in humans by CBZ, approved final version of manuscript. but our results regarding DMO do not support that assumption.

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Brain Stimulation

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Original Article Opposite optimal current ﬂow directions for induction of neuroplasticity and excitation threshold in the human motor cortex

Martin Sommer a,*, Christoph Norden a, Lars Schmack a, Holger Rothkegel a,b, Nicolas Lang a,b, Walter Paulus a a Department of Clinical Neurophysiology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany b Department of Neurology, University Hospital of Schleswig-Holstein Campus Kiel, Germany article info abstract

Article history: Background: Directional sensitivity is relevant for the excitability threshold of the human primary motor Received 8 May 2012 cortex, but its importance for externally induced plasticity is unknown. Received in revised form Objective: To study the influence of current direction on two paradigms inducing neuroplasticity by 9 July 2012 repetitive transcranial magnetic stimulation (rTMS). Accepted 11 July 2012 Methods: We studied short-lasting after-effects induced in the human primary motor cortex of 8 healthy Available online xxx subjects, using 5 Hz rTMS applied in six blocks of 200 pulses each, at 90% active motor threshold. We controlled for intensity, frequency, waveform and spinal effects. Keywords: Transcranial magnetic stimulation Results: Only biphasic pulses with the effective component delivered in an anterioposterior direction Motor cortex (henceforth posteriorly directed) in the brain yielded an increase of motor-evoked potential (MEP) Anisotropy amplitudes outlasting rTMS. MEP latencies and F-wave amplitudes remained unchanged. Biphasic pulses Central sulcus directed posteroanterior (i.e. anteriorly) were ineffective, as were monophasic pulses from either Precentral gyrus direction. A 1 Hz study in a group of 12 healthy subjects confirmed facilitation after posteriorly directed Excitation threshold biphasic pulses only. Plasticity Conclusions: The anisotropy of the human primary motor cortex is relevant for induction of plasticity by subtreshold rTMS, with a current flow opposite to that providing lowest excitability thresholds. This is consistent with the idea of TMS primarily targeting cortical columns of the phylogenetically new M1 in the anterior bank of the central sulcus. For these, anteriorly directed currents are soma-depolarizing, therefore optimal for low thresholds, whereas posteriorly directed currents are soma-hyperpolarizing, likely dendrite-depolarizing and bested suited for induction of plasticity. Our findings should help focus and enhance rTMS effects in experimental and clinical settings. Ó 2012 Elsevier Inc. All rights reserved.

Introduction a coherent picture of how to maximise induced plasticity is still missing puzzle pieces even for the most frequently studied cortical Modulation of human cortical excitability by repetitive trans- site, the motor cortex [6,7]. cranial magnetic stimulation (rTMS) is a promising tool with The human primary motor cortex is sensitive to the direction of a clinical perspective in neuropsychiatry which was first imple- externally induced currents, with currents flowing perpendicular to mented and FDA approved [1] for depression [2,3]. Stimulation the central sulcus in an anterior direction showing the lowest protocols and clinical trials are continuously refined [4,5], but threshold for induction of action potentials measurable in peripheral muscles [8,9]. Whether this anisotropy is relevant for stimulation protocols inducing plasticity is unclear [6,10]. fl This work was supported by the DFG (Deutsche Forschungsgemeinschaft) Traditionally, TMS has been thought to exert its in uence act on grants SO429/2-1 and SO429/2-2 (M.S.), by the Bernstein Center for Computational the crown of the precentral gyrus [11]. The advantage of the Neuroscience grant # 01GQ0432 (W.P.), and the Rose Foundation (W.P.). Part of this presumed stimulation at the crown is its proximity to the skull and work has been previously presented in abstract form (Soc. Neurosci. Abstr. Program to the TMS coil [8]. The disadvantage is the topography of cortical No. 705.12; 2006). columns which are perpendicular to the skull and therefore The authors declare no competing financial interests. * Corresponding author. Tel.: þ49 551 39 66 50; fax: þ49 551 39 81 26. perpendicular to the induced currents, thereby not easy to stimu- E-mail address: [email protected] (M. Sommer). late. To overcome this concern, stimulation of pyramidal tract

1935-861X/$ e see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brs.2012.07.003 2 M. Sommer et al. / Brain Stimulation xxx (2012) 1e8 neurons was thought to occur through stimulation of interneurons eight coil over the dominant motor hand area in healthy young branching more or less horizontally to these cortical columns, and subjects [24,25]. Single suprathreshold pulses applied before, in being in parallel to the skull and thereby easily excitable by the TMS between and after blocks of intervention elicited motor-evoked [12,13]. potentials in a small hand muscle. Their amplitudes and latencies Contrasting with this view, retrograde viral tracer studies in served to control for changes in motor cortex excitability. Inter- monkeys show that even the first-order interneurons which vention consisted of blocks of repetitive subthreshold TMS applied provide monosynaptic input to the pyramidal tract neurons are through the same coil at the same location. located in the anterior bank of the precentral gyrus. Neurons in the crown of the precentral gyrus were only labelled after longer Subjects animal survival times, suggesting more indirect, at least disy- naptic influence on the pyramidal tract neurons (see figure 5 in We investigated 8 healthy subjects (mean age 25.4, range 22e34 [14]). In addition, high-resolution functional imaging studies in years) with no neurological or medical disease and no regular humans indicate movement-related activation deep in the ante- intake of medication except for oral contraceptives in 3 of the 5 rior bank of the central sulcus rather than at the crown of the women. Six subjects were right-handed with an Oldfield [26] precentral gyrus [13]. 10-item score of 100, and two left-handed with a score of 63.0 Hence, an alternative concept has been brought up some years and 38.5. ago, favouring stimulation by TMS of pyramidal tract (PT) cells located in the anterior bank of the central sulcus, tangential to the Methods to quantify excitability skull, rather than to neurons at the crown of the precentral gyrus perpendicular to the skull [13]. EMG recordings of the abductor digiti minimi (ADM) on the side Following this line of thought, a current flow in a posteriore of the dominant hand were recorded as described previously anterior direction passes through the anterior bank of the central [18,22]. Mono- and biphasic TMS pulses were generated by a Mag- sulcus along the physiological flow from layer I to layer VI, and is Pro stimulator (old version, pre-2003) [22] connected to a slightly therefore soma-depolarizing [15,16]. This understanding of current bent figure-of-eight coil (MC B70, Dantec S.A., Skovlunde, flow best explains elementary outcome measures such as motor Denmark). We determined the optimal dominant ADM motor threshold [13]. representation, which is not necessarily identical for all types of As a corollary, pulses from the opposite, i.e. anterioposterior TMS [19], resting motor threshold (RMT) and active motor direction, induce current flow from layer VI to I, are soma- threshold (AMT) as described previously [18,22]. In each subject, hyperpolarizing [15] and likely dendrite-depolarizing. This direc- the optimal motor representation and the threshold were deter- tion resembles that of back propagating action potentials which mined anew for each type of TMS and each direction of stimulation. have been thought to provide a synaptically controlled, associative signal to the dendrites for Hebbian modifications of synaptic Plasticity-inducing protocol at 5 Hz strength [17]. Therefore, we here set out to explore in a systematic way the rTMS was preceded by 3 min of single-pulse TMS at a frequency relevance of current direction not only for the motor threshold, but of 0.25 Hz and at an intensity adjusted to yield baseline MEP also for the induction of lasting changes of cortical excitability. We amplitudes of about 1 mV. Immediately following this baseline, 6 controlled for effects of waveform by using mono- and biphasic blocks of 200 pulses each were delivered at 5 Hz frequency [20]. The pulses, for frequency by using excitatory 5 Hz and inhibitory 1 Hz intensity of 5 Hz stimulation was 90% of the respective AMT indi- stimulation, for intensity using adjusted biphasic pulses, and for vidually determined in each subject, separately for each waveform spinal effects using F-wave amplitudes. The data allow us to and current direction. In between blocks, 15 pulses of baseline conclude that the induction of lasting excitability changes in the intensity were applied at 0.25 Hz frequency. Each interval lasted human motor cortex by externally applied subthreshold stimula- 2 min. After the sixth block, this assessment of corticospinal tion protocols benefits from posteriorly oriented current flow. excitability was prolonged for 7 min at 0.25 Hz. The suprathreshold To accommodate this new view on the TMS site of stimulation, stimuli before and after rTMS and in between rTMS blocks were of we deviate from earlier terminology [18e20] to describe the four the same type and orientation as the rTMS pulses. Sessions were types of rTMS studied here: We name biphasic pulses with effective carried out at least four days apart. current direction in the brain from anterior to posterior “biphasic- P”, and biphasic pulses with effective current direction from Data analysis posterior to anterior “biphasic-A”, and monophasic pulses with current direction from anterior to posterior “monophasic-P” and We normalized peak-to-peak MEP amplitudes to the mean monophasic pulses with current direction from posterior to ante- corresponding baseline and averaged every consecutive 15 MEP rior “monophasic-A”. The current directions given in this manu- amplitudes (1 min) recorded before and after rTMS, and every script always refer to the induced current flow in the brain. For consecutive 15 MEPs obtained in between blocks of 5 Hz stimula- biphasic pulses, we name the second component of the induced tion. MEP amplitudes were entered in a repeated-measures ANOVA current covering the second and third quarter cycles [21,22], with type of rTMS and bin (baseline 1e3, during 1e5, post 1e7) as because it is most relevant [10,23], primarily because of its long within-subject factors. Based on significant main effects, we duration [23]. compared types of rTMS with two-tailed, paired, Fisher-PLSD- corrected t-tests, and we compared each bin of MEPs after rTMS Methods to baseline using paired, two-tailed t-tests.

We compared these four types of rTMS in random order. The Control experiments protocols were approved by the ethics committee of the University Medical Center Göttingen. Written informed consent was obtained For the F-wave control study, we studied three subjects (mean from all participants prior to the experiments. We used standard age 29.5, range 24e35 years; one woman, all with a handedness transcranial magnetic stimulation procedures [20] and a figure- score of 100), two of whom had participated in the main M. Sommer et al. / Brain Stimulation xxx (2012) 1e8 3 experiment. We tested whether the amplitude of F-waves recorded For MEP latency analysis we used the last baseline bin of each from the right ADM were modified by biphasic-P rTMS, adding experiment, i.e. the last 15 baseline MEPs from the main experi- electrical stimuli (Multipulse Stimulator, Model D 185, Digitimer, ment and the last 18 baseline MEPs from the 1 Hz frequency Welwyn Garden City, UK) through a conventional EMG stimulation experiment. These latencies were analyzed using a repeated- block over the ulnar nerve at the wrist [27] to the set-up described measures ANOVA with type of TMS (4 levels) as within-subject above. Blocks of 20 F-waves were obtained with 0.25 Hz repetition factors. In addition, we performed paired, two-tailed t-tests on frequency before and three times after the rTMS intervention, MEP latencies from the biphasic-P type of TMS. For the main which was identical to the principal experiment. In addition, we experiment (n ¼ 8), we compared latencies from the third bin after obtained blocks of 45 MEPs (3 min) from biphasic-P pulses before rTMS, where the maximum lasting facilitation was observed, to the and twice after rTMS, adjusted in intensity to yield baseline MEP last baseline. For the 1 Hz frequency experiment (n ¼ 12), we amplitudes of w1 mV, at a repetition frequency of 0.25 Hz. After compared the MEP latencies of the 4th bin after rTMS to the last rTMS, recordings were intermingled (post 1 block of F-waves, post baseline bin. 1 block of MEPs, post 2 block of F-waves and so forth). We described For correlation analysis, we correlated RMT and MEP latency alterations from baseline for the amplitudes from MEPs and from across all data from the main experiment and the 1 Hz frequency F-waves. experiment using Pearson’s correlation coefficient and using linear To dissociate effectiveness for threshold and effectiveness for regression ANOVA. KolmogoroveSmirnov tests confirmed normality inducing plasticity, we conducted an intensity control study.We of all datasets, the level of significance was set at P < 0.05. repeated the main experiment twice in a group of 5 healthy young subjects (mean s.d., age, 24.6 3.0; handedness, 71.0 9.75, three women). In random order, we either used biphasic-P pulses Results with an intensity as in experiment 1 (90% of AMT of biphasic-P pulses), or we reduced the intensity to the equivalent of 90% of Plasticity-inducing protocol at 5 Hz AMT of biphasic-A pulses determined individually in each subject. We analyzed this using a repeated-measures ANOVA with intensity We observed larger MEP amplitudes only after biphasic-P pul- (2 levels) and bin (baseline 1e2, post 1e7) as within-subject factors. ses. This facilitation persisted on average for 5 min after rTMS before returning to baseline values (Fig. 1). The other three types of rTMS did not modify MEP amplitudes significantly during or after Comparator study with 1 Hz stimulation frequency stimulation (P ¼ 0.0004, effect of bin, ANOVA; P ¼ 0.030, interaction of type by bin, ANOVA). Post-hoc t-tests yielded a significant To control for the influence of rTMS frequency, we expanded an difference between biphasic-P pulses and all other types of rTMS. earlier set of 1 Hz rTMS data, part of which has been published Only for the biphasic-P pulses did bins post 3 and post 5 differ from previously [18]. We studied 12 healthy controls (7 men, 5 women; baseline (P < 0.05, paired, two-tailed t-tests, Table 1). mean age 25.3, range 19e31 years) four times, using the same For the effective biphasic-P type of rTMS, MEP latencies stimulator, coil and rTMS pulse types as in the main experiment. remained unchanged at bin ‘post 30 (mean s.d.; 22.98 1.03 ms) The rTMS intervention consisted of 900 pulses at 1 Hz frequency and 90% RMT intensity. Single-pulse TMS at 0.1 Hz with an intensity adjusted to yield MEP amplitudes of about 1 mV was used for 15 min before the rTMS intervention to detect a baseline of excitability, and for 30 min after rTMS to detect lasting effects. We subdivided the rTMS intervention into five blocks of 180 pulses each, separated by pauses of 2 min duration. During each pause we applied 10 pulses of baseline intensity at 0.1 Hz frequency. We normalized all peak-to-peak MEP amplitudes to the mean corresponding baseline and averaged the amplitudes of every consecutive 18 MEPs (3 min) recorded before and after rTMS, and every bin of suprathreshold pulses during rTMS. For effects during rTMS, we used a repeated-measures ANOVA for the normalized MEP amplitudes in between rTMS blocks with ‘type of TMS’ (4 levels) and ‘time’ (baseline 1e5 and during 1e4) as within-subject factors. For lasting effects, we entered the normalized MEP amplitudes of the first four bins after rTMS in a repeated-measures ANOVA with ‘type of TMS’ (4 levels) and ‘time’ (baseline 1e5 and post 1e4) as within-subject factors. Based on significant main effects, we compared types of rTMS with two-tailed, paired, Fisher PLSD-corrected t-tests, and we compared each bin of MEPs after rTMS to baseline using paired, two-tailed t-tests.

Analysis of resting motor threshold and latency

We pooled RMT values and MEP latencies from all 8 subjects of the main experiment and the 12 subjects from the 1 Hz frequency Figure 1. Current direction-dependent plasticity at 5 Hz. MEP amplitudes before, in experiment. RMT was analysed using a repeated-measures ANOVA between and after six blocks of rTMS of 200 pulses each at 90% AMT; four types of with type of TMS (4 levels) as within-subject factors. rTMS studied in 8 subjects. Note that lasting facilitation is observed with one TMS type only, biphasic-P pulses (P < 0.030, interaction of bin by type of rTMS, ANOVA). Additionally, AMT values from all 8 subjects of the main Mean SEM. Asterisks indicate post-hoc t-tests vs. baseline, P < 0.05. Dotted line experiment were analyzed using a repeated-measures ANOVA with indicates the unconditioned baseline amplitude. Vertical light bars indicate blocks of type of TMS (4 levels) as within-subject factors. subthreshold rTMS. 4 M. Sommer et al. / Brain Stimulation xxx (2012) 1e8

Table 1 Table 2 Main experiment with 5 Hz rTMS. Intensity control experiment.

Source of variation DF Sum of Mean F value P value Source of variation DF Sum of Mean F value P value squares square squares square Subject 7 10.26 1.47 Subject 4 42071.17 10517.79 Type of rTMS 3 6.27 2.09 3.095 0.049 Type of rTMS 1 77.96 77.96 0.01 0.9261 Type of rTMS subject 21 14.18 0.68 Type of rTMS subject 4 32027.46 8006.87 Bin 14 6.09 0.44 3.174 0.0004 Bin 8 30438.62 3804.83 1.922 0.0908 Bin subject 98 13.42 0.14 Bin subject 32 63334.32 1979.20 Type of rTMS bin 42 7.93 0.19 1.501 0.0298 Type of rTMS bin 8 1905.04 238.13 0.17 0.9935 Type of rTMS bin subject 294 37.01 0.13 Type of rTMS bin subject 32 44891.47 1402.86

Results of repeated measures ANOVA speciﬁed in the methods section. DF ¼ degrees Results of repeated measures ANOVA speciﬁed in the methods section. DF ¼ degrees of freedom. of freedom. as compared to baseline (22.86 1.06 ms; P ¼ 0.34, paired, two- In between blocks of 1 Hz rTMS, we observed a uniform inhi- tailed t-test). bition of MEP amplitudes (P < 0.0001, effect of time, ANOVA, no effect of type of rTMS, no interaction; Table 4). Control experiment: F-waves Motor threshold and MEP latency In a subsample of 3 subjects, a control study did not show any increase of F-wave amplitudes, excluding any relevant contribution Motor threshold was lowest and MEP latency shortest with of spinal excitability. While MEP amplitudes increased from biphasic-A pulses; motor threshold was highest and latency longest 1.07 mV (SD 0.07) at baseline to 1.56 mV (SD 0.69) at post 1 and with monophasic-P pulses. The motor threshold depended on the 1.66 mV (SD 0.70) at post 2, F-wave amplitudes decreased from pulse type (P < 0.0001, effect of type, ANOVA; Fig. 4A, Table 5a), as 0.17 mV (SD 0.14) at baseline to 0.14 mV (SD 0.10) at post 1, 0.12 mV did the MEP latency (P < 0.0001, effect of type, ANOVA, Table 5b), as (SD 0.08) at post 2 and 0.13 mV (SD 0.11) at post 3. did AMT in the main experiment (P < 0.0001, effect of type, ANOVA, Table 5c). Across all types of TMS, RMT and MEP latency were Control experiment: stimulus intensity positively correlated (P ¼ 0.011, ANOVA, Fig. 4B).

In another subsample of 5 subjects, adjusting the intensity of the effective, biphasic-P type to the lower intensity of the ineffective, Adverse events biphasic-A type did not abolish the facilitation (Fig. 2) and did not yield a signiﬁcant effect of intensity (P > 0.9, ANOVA, Table 2). No adverse events were reported by the subjects or observed by the investigators. Comparator study with 1 Hz stimulation frequency

A comparator study exploring the direction-sensitivity of rTMS protocols at 1 Hz stimulation frequency in a distinct sample of 12 healthy subjects yielded dissociated effects after posteriorly directed currents: Biphasic-P pulses induced a delayed facilitation of MEP amplitudes, whereas monophasic-P pulses induced a lasting inhibition of MEP amplitudes. By contrast, anteriorly directed currents did not result in any signiﬁcant lasting effects (P ¼ 0.049, effect of type of rTMS, ANOVA, Fig. 3, Table 3). Post-hoc tests revealed a signiﬁcant difference between these two types of rTMS (P < 0.05, paired, two-tailed t-tests).

Figure 3. Current direction-dependent plasticity at 1 Hz. MEP amplitudes in between and after ﬁve blocks of 180 rTMS pulses each at 1 Hz and 90% RMT intensity; four types of rTMS studied in 12 subjects. Only biphasic-P directed pulses yield a delayed facilitation after rTMS, while only monophasic-P pulses yield an inhibition (P ¼ 0.049, effect Figure 2. Intensity control experiment. MEP amplitudes in ﬁve subjects after biphasic- of type of rTMS, ANOVA). These lasting effects differ from observations during rTMS P rTMS with normal intensity (90% AMT of biphasic-P pulses) or lowered intensity blocks where all TMS types yield an inhibition (P < 0.001, effect of time, ANOVA). (stimulus output as for 90% AMT of biphasic-A pulses). Mean SEM. Reducing the Mean SEM. Asterisks indicate post-hoc t-tests vs. baseline, P < 0.05. Dotted line intensity does not abolish the effect. Dotted line indicates the unconditioned baseline indicates the unconditioned baseline amplitude. Vertical light bars indicate blocks of amplitude. subthreshold rTMS. M. Sommer et al. / Brain Stimulation xxx (2012) 1e8 5

Table 3 A 1 Hz rTMS comparator study, effects after rTMS.

Source of variation DF Sum of Mean F value P value squares square Subject 11 0.88 0.08 Type of rTMS 3 1.19 0.40 2.904 0.0493 Type of rTMS subject 33 4.50 0.14 Bin 8 1.02 0.13 2.117 0.0423 Bin subject 88 5.30 0.06 Type of rTMS bin 24 2.05 0.09 1.325 0.1464 Type of rTMS bin subject 264 17.04 0.07

Results of repeated measures ANOVA speciﬁed in the methods section. DF ¼ degrees of freedom.

Discussion B Here we report that plastic after-effects of subthreshold rTMS depend on current flow direction, but with a posterior direction, opposite to that long known to provide the lowest corticospinal tract excitation threshold by anterior current flow. This finding holds true particularly for the facilitatory plasticity at 5 Hz and is less evident for the inhibitor plasticity at 1 Hz. Optimizing current flow direction will likely help enhance the efficacy of rTMS applications in neuropsychiatry [6,7,28].

Analogy to slice studies

The observation that opposite current directions are optimal for either initiating action potentials or for inducing plasticity is reminiscent of orthodromic vs. antidromic stimulation established in slice preparations [29]. Currents flowing orthodromically [29] from the apical dendritic tree towards the soma are “soma-depolarizing” [15,16]. They enhance the population spike amplitude recorded from a hippocampal slice [15], have a low excitation Figure 4. Excitation threshold and motor-evoked potential latency. Mean SEM, data from 5 Hz (n ¼ 8) and 1 Hz (n ¼ 12) experiments did not differ significantly and were threshold for inducing a population spike [15,30], and they move pooled. (A) Resting motor threshold at baseline for each type of TMS (P < 0.0001, effect the initiation site of the population spike further downstream of type, ANOVA). (B) Correlation analysis of resting motor threshold and MEP latency, towards the soma, at least in some circumstances [30]. By contrast, across all data from both frequencies and all TMS types (P ¼ 0.011, ANOVA). The currents flowing from the soma towards the apical dendritic tree equation describes the linear trend line with the adjacent correlation coefficient, are soma-hyperpolarizing [15] and do not facilitate the population indicating that higher RMT values are correlated with longer MEP latencies. spike amplitude [15,30]. With regard to plasticity, repeated depolarization of the dendrite by subthreshold posteriorly directed TMS assumed that specific connections to the PT cells are the explana- in the absence of somatic output can be assumed to drive the tion for directional selectivity. If the main TMS effects on the human dendrite and to make it more susceptible to the input from cortical primary motor cortex are induced in the anterior bank of the and subcortical afferents, and to back-propagating action poten- central sulcus and not at its gyral crown, i.e. in the “new” oligosy- tials, which enhance plasticity [17]. naptic M1 rather than in the “old” polysynaptic M1 [14], then a current flow parallel to the orientation of PT cells can be assumed, Cortical topography resulting in a true anti- or orthodromic current flow. Higher TMS field intensity at the crown with tangential current flow is probably Transferring these slice results to TMS data is facilitated by outweighed by perpendicular current flow in the sulcus. This was fl assuming a tangential current ow in the wall of M1 targeting the confirmed in some [10], though not in all models [31]. This is also in new M1. If the assumption is true as summarized in [13] and further line with anatomical data in humans with regard to the existence of “ ” discussed in [10], that the new M1 contains the monosynaptic two different groups of Betz cells, one group located in the depth of pyramidal cells to the spinal cord [14], then it no longer need be the central sulcus next to area 3a and the other more rostrally at the junction with area 6 [32], see also [33]. At least the former should be better targeted by a tangential current flow with the reference at Table 4 1 Hz rTMS comparator study, effects during TMS.

Source of variation DF Sum of Mean F value P value Table 5a squares square RMT of main experiment and 1 Hz comparator study pooled. Type of rTMS 3 0.36 0.12 0.6 0.6183 Subject 44 8.78 0.20 Source of variation DF Sum of squares Mean square F value P value Bin 8 15.47 1.93 24.326 <.0001 Subject 19 9509.55 500.50 Bin type of rTMS 24 1.35 0.06 0.708 0.8444 Type of TMS 3 15855.27 5285.09 215.3 <.0001 Bin subject 352 27.99 0.08 Type of TMS subject 57 1399.21 24.55

Table 5b their ability to induce plastic effects [37,38]. On a network level, the MEP latency of main experiment and 1 Hz comparator study pooled. different pulse shapes could target different cortical layers [39] and Source of variation DF Sum of squares Mean square F value P value different fibres [7], such as cortical or subcortical afferents. The Subject 19 121.65 6.40 configuration of the biphasic pulse with its oppositely directed Type of TMS 3 44.86 14.95 25.39 <.0001 components is advantageous in inducing network effects by dual Type of TMS subject 57 33.57 0.59 activation of different [40] and possibly remote [41] inputs to Results of repeated measures ANOVA specified in the methods section. DF ¼ degrees a given pyramidal tract neuron and its dendritic tree [42].For of freedom. example, in the rat motor cortex, tetanizing pathways in layer I and in layer II/III resulted in more facilitation than tetanizing either of them alone [40], and in the rat hippocampus, the converging effect the contralateral forehead. Following this line of thought, anteriorly of coactivation depends on the degree of spatial overlap of the directed currents excite cortical columns in the anterior bank terminal fields of the pathways, or the involvement of crossing orthodromically, in a soma-depolarizing fashion, which is optimal pathways [42]. for inducing action potentials of pyramidal tract neurons. Following this view, the lack of facilitation after biphasic-A rTMS Conversely, posteriorly directed currents excite cortical columns is presumably related to the short duration of the first quarter cycle antidromically, being soma-hyperpolarizing and dendrite- running antidromically. The situation is different for supra- depolarizing, and therefore enable plastic changes in dendrite threshold rTMS, where biphasic pulses of either direction induce excitability to occur in the absence of corticospinal output. modest facilitation during rTMS [19]. This hypothetical explanation is corroborated (a) by the latency differences we observed here and (b) by earlier results with I waves. Comparison with other data First, the correlation of latency and excitation threshold we found in humans (Fig. 4B) confirms that action potentials are initiated at Our data expand the findings of Quartarone and colleagues [43]. different sites by different TMS pulses [34]. The site of initiation is They showed an rTMS-induced facilitation with biphasic-P pulses more downstream of the cortical column with low-threshold TMS using a Magstim Rapid stimulator, but measured after-effects with types and shorter latencies, but more upstream and possibly in the a Magstim 200, producing monophasic-P pulses. The facilitation dendritic tree for TMS types with high thresholds and longer was intensity-dependent, because it was present at the high 90% latencies, particularly with regard to monophasic-P pulses. Second, RMT and absent at the low 90% AMT. the I wave patterns observed in epidural recordings of corticospinal Our data also expand a report on 10 Hz rTMS over the primary volleys induced by TMS pulses over the motor cortex further motor cortex by Arai and colleagues [44]. Using 10 blocks of 100 support this assumption, because anteriorly directed monophasic pulses each, these authors studied biphasic-A pulses and or biphasic pulses induce early I waves, whereas monophasic-P monophasic-A pulses, with two different intensities of 90% AMT pulses yield later I waves, at least in some subjects [34]. Possibly, and 90% RMT. Consistent with our data, no significant MEP changes the high-threshold posteriorly directed currents achieve action were found with 90% AMT intensity. At 90% RMT, the authors report potentials only by activating interneurons within the dendritic tree a lasting facilitation that was stronger after monophasic-A pulses [34]. Studies on short afferent inhibition also suggest different than with biphasic-A pulses. This is not consistent with our current neuronal mechanisms activated by monophasic-P as compared to findings obtained with slower frequencies and fewer stimuli. monophasic-A TMS [35]. Rather, their finding is reminiscent of the strong facilitation we reported with monophasic-A pulses during suprathreshold 5 Hz rTMS [19]. When inhibition and when facilitation? Other cortical areas may require different current orientations to modulate cortical excitability in a lasting manner. E.g., MEP facili- In our data, inhibition was only present after 1 Hz rTMS, while tation using an rTMS protocol similar to ours (5 blocks of 150 pulses facilitation was observed after 1 Hz and after 5 Hz rTMS, suggesting at 5 Hz) was achieved with biphasic latero-medially oriented pulses that frequency plays a clearer role for inhibition than for facilitation. over the supplementary motor area SMA [45]. Regarding waveform, inhibition was better induced by monophasic The rTMS frequency has been considered for a long time to be pulses, but facilitation was better induced by biphasic pulses. the single most important factor to determine whether a train of Earlier data also suggest that the pattern of stimulation, especially rTMS leads to an increase or a decrease of corticospinal excitability. the introduction of intervals plays a role, too [4,5,20]. When 1 Hz has been regarded the gold standard of a watershed for subthreshold 5 Hz rTMS was applied without intervals it was differentiating between inhibition and facilitation by many inhibitory and not facilitatory, as expected [20]. researchers with rTMS-frequencies at or below 1 Hz leading to Cellular, stimulation site and network influences probably decreased corticospinal excitability and higher rTMS frequencies contribute to the direction of induced plasticity. On a cellular level, leading to an increase of corticospinal excitability and thus facili- it is conceivable that different cell and fibre types with different tation of motor evoked potentials. However, 1 Hz rTMS can also be membrane properties are influenced differently by different pulse facilitatory and 5 Hz rTMS can be inhibitory depending on the state shapes [36]. Regarding stimulation site, the more upstream site of of cortical excitability at time given [46,47]. This indicated that the action of dendrite depolarizing pulses [13] likely contributes to ‘classical’ view of pure frequency-dependence of rTMS effects may be too simple. This is again confirmed by the data presented in this Table 5c manuscript. AMT from main experiment.

Source of variation DF Sum of squares Mean square F value P value Stimulus intensity differences as a potential confounding factor Subject 7 841.62 120.23 Type of TMS 3 4749.15 1583.05 50.574 <.0001 For biphasic-P pulses, the intensity control experiment did not Type of TMS subject 21 657.33 31.30 give any indication of a pivotal relevance of intensity among Results of repeated measures ANOVA specified in the methods section. DF ¼ degrees biphasic pulses (Fig. 2). Compared to the monophasic pulses of freedom. studied here, the effective biphasic-P pulses had a lower motor M. Sommer et al. / Brain Stimulation xxx (2012) 1e8 7 threshold and rTMS stimulus intensity. In other words, we found propagating and dendrite-depolarizing, and thus best suited for monophasic conditions with higher intensities to be less effective in induction of plasticity. Our findings should help to focus and inducing facilitation. enhance rTMS effects in future experimental and clinical settings. With regard to inhibition outlasting rTMS, however, we cannot rule out a stimulus intensity confound, because monophasic-P Acknowledgements pulses do have the highest motor threshold and therefore allow the highest intensities to be applied to the brain without reaching We thank Michael Nitsche, Dirk Czesnik, Asif Rahman and motor threshold, which is possibly related to their site of action Alexander Opitz for discussions. upstream of the cortical column suggested by their long latencies. These high intensities likely, though speculatively, excite back- References propagating and bending parts of the dendritic tree and its afferent interneurons. The initial quarter cycle of monophasic-P [1] Melkerson MN. Food and drug administration regulation on NeuroStar TMS therapy system. www.accessdatafda.gov/cdrh_docs/pdf8/K083538.pdf pulses is higher in amplitude, allowing for a larger charge transfer 2008:downloaded November 8, 2009. in that period. 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Research report Right-shift for non-speech motor processing in adults who stutter

Nicole E. Neef a,b, Kristina Jung c, Holger Rothkegel a,b, Bettina Pollok d, Alexander Wolff von Gudenberg c, Walter Paulus a,b and Martin Sommer a,b,* a Department of Clinical Neurophysiology, Georg-August-University Goettingen, Germany b Center for Systems Neuroscience, Goettingen, Germany c Institut der Kasseler Stottertherapie, Bad Emstal, Germany d Institute of Clinical Neuroscience and Medical Psychology, Heinrich-Heine-University Duesseldorf, Germany article info abstract

Article history: Introduction: In adults who do not stutter (AWNS), the control of hand movement timing is Received 5 November 2009 assumed to be lateralized to the left dorsolateral premotor cortex (PMd). In adults who Reviewed 24 April 2010 stutter (AWS), the network of speech motor control is abnormally shifted to the right Revised 24 April 2010 hemisphere. Motor impairments in AWS are not restricted to speech, but extend to non- Accepted 2 June 2010 speech orofacial and finger movements. We here investigated the lateralization of finger Action editor Henry Buchtel movement timing control in AWS. Published online xxx Methods: We explored PMd function in 14 right-handed AWS and 15 age matched AWNS. In separate sessions, they received subthreshold repetitive transcranial magnetic stimulation Keywords: (rTMS) for 20 min at 1 Hz over the left or right PMd, respectively. Pre- and post-stimulation Persistent developmental stuttering participants were instructed to synchronize their index finger taps of either hand with an Repetitive transcranial magnetic isochronous sequence of clicks presented binaurally via earphones. Synchronization stimulation accuracy was measured to quantify the effect of the PMd stimulation. Dorsolateral premotor cortex Results: In AWNS inhibition of left PMd affected synchronization accuracy of the left hand. Compensatory mechanism Conversely, in AWS TMS over the right PMd increased the asynchrony of the left hand. Conclusions: The present data indicate an altered functional connectivity in AWS in which the right PMd seems to be important for the control of timed non-speech movements. Moreover, the laterality-shift suggests a compensatory role of the right PMd to successfully perform paced finger tapping. ª 2010 Elsevier Srl. All rights reserved.

1. Introduction malfunctioning in developmental stuttering (Brown et al., 2005; Fox et al., 1996; Ludlow and Loucks, 2003). Stuttering is Fluent speech requires the well timed selection, initiation, characterized by an impairment of speech rhythm or ﬂuency execution and monitoring of motor sequences. The relevant (Bloodstein and Ratner, 2008). Speech disruptions typically cortical and subcortical neural systems appear to be include blocks, repetitions, or prolongations of speech

* Corresponding author. Department of Clinical Neurophysiology, University of Goettingen, Robert-Koch-Str. 40, D-37075 Goettingen, Germany. E-mail address: [email protected] (M. Sommer). 0010-9452/$ e see front matter ª 2010 Elsevier Srl. All rights reserved. doi:10.1016/j.cortex.2010.06.007

Please cite this article in press as: Neef NE, et al., Right-shift for non-speech motor processing in adults who stutter, Cortex (2010), doi:10.1016/j.cortex.2010.06.007 2 cortex xxx (2010) 1e10

segments (WHO, 2007b), and may be accompanied by move- adequate length and drives motor commands at the end of ments of face and limb muscles and by negative emotions each interval. The original WingeKristofferson model was such as fear or embarrassment. About 5% of the population concerned with the special case of self-paced finger tapping stutters at some point during childhood (Mansson, 2000). and therefore neglected the process of integrating external Although spontaneous recovery rate is high, stuttering cues. This contrasts with finger tapping in synchrony with an without obvious neurological origin persists after puberty in acoustically presented pacer, a timed motion task that addi- about 1% of adults (Andrews and Harris, 1964; Bloodstein and tionally involves the integration of the external event and the Ratner, 2008; Craig et al., 2002). Exploring the underlying monitoring of the synchrony of the pacer and the tapping. neural mechanisms of this disorder provides insights into Finger tapping accuracy can be disturbed by transcranial mechanisms of dysfluent speech production and into models magnetic stimulation (TMS) (Doumas et al., 2005; Levit- of speech planning and production in general. These insights Binnun et al., 2007; Malcolm et al., 2008; Pollok et al., 2008), into the physiology of stuttering may ultimately serve to a neurophysiological technique inducing a brief electric improve treatments enhancing speech fluency. current in the brain using a magnetic field to pass the scalp Temporal patterns in speech occur on multiple timescales and the skull safely and painlessly. Repetitive TMS (rTMS) is (i.e., subsegmental, segmental and suprasegmental, Levelt, capable of inducing excitability changes of neural networks 1989). In adults who stutter (AWS), acoustic-temporal and outlasting the stimulation period (Hallett, 2000; Miniussi et al., spatio-temporal characteristics are affected in stuttered and 2008; Siebner and Rothwell, 2003; Siebner et al., 2009), thereby fluent speech on all these timescales (Jancke, 1994; Kleinow temporarily disrupting activity in local or remote cortical and Smith, 2000; Max and Gracco, 2005; Prins and Hubbard, areas (Wagner et al., 2009; Walsh and Rushworth, 1999). Thus, 1992). Most consistent are the observations of increased rTMS disrupts brain functions for a finite time with relatively variability of duration and relative timing of acoustic and high spatial resolution. kinematic features. Additionally, stuttering has been associ- In the present study rTMS was employed to induce a tran- ated with altered auditory feedback control mechanisms (Max sient virtual lesion of the dorsolateral premotor cortex (PMd). et al., 2004; Tourville et al., 2008). Altogether, these facts Traditionally the premotor cortices (PM) were assumed to be underline a deficit of speech motor timing and the impact of key structures in the motor domain and thereby associated the timing of auditory information during speaking in AWS. with the preparation and the organization of movements and Alterations of timing abilities in AWS exceed the domain of actions (Wise, 1985). Imaging studies suggest a specific speech and affect the motor control of non-speech move- significance of the PMd for cognitive functions (Abe and ments as well. For example, AWS performed poorly in repro- Hanakawa, 2009), sensorimotor integration (Pollok et al., ducing varying rhythmic patterns (Hunsley, 1937)or 2009; Schubotz et al., 2003) and rhythm perception unpredictable digit sequences (Webster, 1986). Additionally, (Bengtsson et al., 2009), as well. Recent studies provide AWS exhibit prolonged initiation and execution times in evidence for a specific role of the left PMd for movement finger movement sequencing tasks (Smits-Bandstra et al., timing of both hands (Pollok et al., 2009, 2008). Interestingly, 2006; Webster, 1997) and increased manual reaction times externally paced finger movements as well as syllable repeti- (Bishop et al., 1991; Webster and Ryan, 1991). Phase variability tion seem to recruit the same cerebral network involving the is greater during bimanual coordination of auditory paced left PMd (Riecker et al., 2006). However, the PMd seems to play movements (Zelaznik et al., 1997) and movement variability is a role during fluency enhancing mechanisms in AWS. Fluency increased during simultaneous synchronization of speech is reliably enhanced when speech is timed to a pacer: either an and hand movements (Hulstijn et al., 1992). However, studies external pacer such as a rhythmic beat (Wingate, 2002; Wohl, on auditory paced isochronous finger movements did not find 1968), the unison speaking with another person (Adams and differences of timing accuracy and timing variability between Ramig, 1980; Ingham and Carroll, 1977; Saltuklaroglu et al., AWS and controls (Hulstijn et al., 1992; Max and Yudman, 2009), or an internal pacer such as rhythmic arm swinging or 2003; Melvine et al., 1995; Zelaznik et al., 1994). a finger tapping (Bloodstein and Ratner, 2008). Alternative Two separate processes have been related to timing accu- fluency enhancing techniques are delayed or frequency shif- racy: a neural clock mechanism (Rao et al., 1997; Ivry and ted auditory feedback (Antipova et al., 2008; Van Riper, 1970). Spencer, 2004), and an emergent property of the kinematics Such fluency enhancing mechanisms involve right premotor of movements itself (Ivry and Spencer, 2004; Mauk and regions as well as the cerebellum (Braun et al., 1997; Fox et al., Buonomano, 2004). This dissociation between event timing 1996; Tourville et al., 2008; Watkins et al., 2008). Hence, the PM and emergent timing has been corroborated by previous find- seem to play an important role for motor timing control as ings (Spencer et al., 2003; Zelaznik et al., 2005, 2002). Timing in well as the implementation of fluency enhancing techniques. the sub- and supra-second range involves dissociable neural Theoretical frameworks on stuttering suggest an aberrant networks (Gibbon et al., 1997; Lewis and Miall, 2003; Wiener timing of neural activity in different brain regions that are et al., 2010). Sub-second timing engages cerebello-thalamo- relevant for speech processing (Alm, 2004; Howell, 2004; cortical network (Pollok et al., 2005), whereas supra-second Ludlow and Loucks, 2003). Specifically, the basal ganglia- timing tasks were more prone to activate cortical structures cortical route might be impaired in providing internal cues for such as supplementary motor area (SMA) and prefrontal cortex the exact timing of movements, while the PMd in concert with (Wiener et al., 2010). For an event timing task like self-paced the cerebellum successfully utilizes external time cues finger tapping, Wing and Kristofferson (1973) indicate resulting in enhanced fluency for example during metronome a dichotomy between central clock and motor execution by speaking (Alm, 2004). Interestingly, in AWS even a non-speech suggesting that a central timekeeper supplies intervals of the motor task like externally paced finger tapping mirrored an

Please cite this article in press as: Neef NE, et al., Right-shift for non-speech motor processing in adults who stutter, Cortex (2010), doi:10.1016/j.cortex.2010.06.007 cortex xxx (2010) 1e10 3

irregular right-shifted activation (Morgan et al., 2008). This female] participated in this study. Table 1 contains details of increased right pre-central activation suggests that the cortical the participants. Stuttering participants were recruited from contribution to the process of timed movements is less left the Stuttering self-help group of Goettingen and the Institute lateralized. The present study aims at further investigating the for the Kassel Stuttering Therapy. Three AWS had already assumption of a hemispheric shift of motor functions in AWS taken part in an earlier TMS study (Sommer et al., 2009). The by means of an induced virtual lesion of the left and right PMd groups were matched and statistics did not yield any group in AWS and adults who do not stutter (AWNS). differences for age (T ¼ .65, p ¼ .5), handedness (Oldﬁeld, 1971; Z ¼.73, p ¼ .46) and level of education (Z ¼1.28, p ¼ .2), amount of musical training and gender. AWS produced

2. Methods significantly more stuttered syllables than AWNS [meanAWS ¼ < 9.0 8.0 (SD), meanAWNS .6 .4 (SD); Z 4.6; p .001; for 2.1. Participants details on statistics see data analysis section]. Stuttering severity was very mild in five, mild in three, moderate in two, Fourteen right-handed AWS [mean age 30.3 11.4 (SD); one severe in two and very severe in two AWS according to the female] and fifteen AWNS [mean age 28.1 5.0 (SD); one Stuttering Severity Index (SSI-3). Inter-rater reliability analysis

Table 1 e Characteristics of participants.

Age Gender Education Instrument Handedness Mother AMT AMT Suttered SSI-3 Age of tongue right left syllables score stuttering FDI FDI onset

AWS 38 m 6 Yes 100 G 39 42 3.1 17 3.5 27 m 6 Yes 100 G 43 36 6.9 21 6 21 m 3 No 100 G 45 50 1.9 8 2.5 44 m 2 Yes 60 G 57 54 2.9 14 2 42 m 6 No 70 G 38 40 25.2 33 5 18 m 3 No 90 G 49 44 23.8 43 4.5 18 m 1 Yes 80 G 68 73 16.3 40 4 28 m 6 Yes 90 K 54 57 9.8 28 4.5 33 m 6 No 70 T 44 57 3.8 27 2.5 19 f 2 Yes 70 G 63 46 4.5 23 2.5 54 m 1 No 75 G 57 63 1.5 7 4 28 m 6 Yes 30 G 38 36 16.5 32 4.5 36 m 2 No 70 G 63 68 3.2 16 5 18 m 1 Yes 100 G 51 59 5.9 18 6

Median 28.0 3 77.5 50.0 52.0 5.2 22.0 4.3 Mean 30.3 79.0 50.6 51.8 9.0 23.4 4.0 SD 11.4 19.8 10.0 11.7 8.2 11.0 1.3

AWNS 29 m 5 No 60 G 40 43 .4 25 m 3 No 100 G 48 54 1.5 39 m 6 No 57 G 50 54 1.1 34 m 6 No 100 G 55 58 .9 23 m 4 Yes 100 G 41 40 .3 27 f 6 No 80 H 28 30 .5 31 m 6 Yes 70 I 48 41 .5 33 m 6 No 90 G 47 50 .4 30 m 6 No 63 G 46 42 .2 20 m 3 Yes 70 G 56 56 .3 25 m 3 Yes 60 G 38 50 .8 24 m 3 Yes 50 G 51 47 .3 24 m 4 Yes 60 G 57 57 .5 31 m 3 No 80 G 52 40 .3 27 m 5 No 100 G 42 40 .8

Median 27.0 5 70.0 48.0 47.0 .5 Mean 28.1 76.0 46.6 46.8 .6 SD 5.0 18.1 7.8 8.15 .4

Test ( p) T ¼ .65 (.5) Z ¼1.28 (.2) Z ¼.73 (.46) F ¼ 1.87 (.18) Z ¼4.6 (<.001)

AWS ¼ adults who stutter; m ¼ male, f ¼ female; SD ¼ standard deviation; AMT ¼ active motor threshold; FDI ¼ ﬁrst dorsal interosseous; G ¼ German, K ¼ Kannada, T ¼ Turkish, H ¼ Hungarian, I ¼ Italian; level of education was estimated as follows: 1 ¼ school, 2 ¼ high school, 3 ¼ less than 2 years college, 4 ¼ 2 years college, 5 ¼ 4 years college, 6 ¼ postgraduate; handedness was quantiﬁed with the 10-item scale of the Edinburgh Handedness Inventory; stuttered syllables were mean percentage out of not less than 340 read and 500 spoken syllables.

Please cite this article in press as: Neef NE, et al., Right-shift for non-speech motor processing in adults who stutter, Cortex (2010), doi:10.1016/j.cortex.2010.06.007 4 cortex xxx (2010) 1e10

yielded an unjust intra-class correlation coefficient (ICCunjust) presented binaurally via dynamic, closed-ear headphones of .94 (95% CI .82 -.98) and intra-rater reliability analysis yiel- (Sennheiser HD 280; up to 32 dB attenuation of outside noise). ded an ICCunjust of .97 (95% CI .81 - .98). Click intensity was individually adjusted to a level perceived as None of the participants had a self-reported history of loud by the participants. The pacing signal was triggered and speech, language or hearing problems, with the exception of the onsets of space bar presses were recorded by using Eprime stuttering in AWS. According to the definition (WHO, 2007a) (http://www.pstnet.com). We quantified performance by cluttering was recognized by rapid, erratic, and dysrhythmic calculating (1) the asynchrony, the averaged temporal distance speech dysfluency with distinct speech timing abnormalities. between the onset of the pacing signal and finger taps, and (2) On this ground we excluded one fifteenth putative participant the inter-tap interval (ITI)-variability, the variation of the time who exhibited both stuttering and cluttering. None of the between two consecutive taps. participants showed neurological or medical abnormalities on routine examination. None of the participants were taking 2.4. Stimulation technique drugs affecting the central nervous system at the time of the study. The local Ethics Committee approved the study and all TMS was applied while participants sat comfortably in participants gave written informed consent according to the a reclining chair. A figure-8-shaped stimulation coil connected declaration of Helsinki. to a Magstim rapid2 stimulator (Magstim Company, Dyfed, Wales, UK) was positioned tangentially to the scalp with the 2.2. Fluency assessment handle pointing backwards and rotated away from the midline by 45. The junction of the two wings of the figure-8-coil was The fluency assessments were performed and independently held flat on the skull. The pulse configuration was biphasic analyzed by a qualified speech-language pathologist (N.N.) with an initial posterioreanterior current flow in the brain. The and a qualified clinical linguist (K.J.). In compliance with the motor hot spot was localized at the optimal point for eliciting German version of the SSI-3 (Sandrieser and Schneider, 2008), motor evoked potentials (MEPs) in the contralateral first dorsal speech samples of all participants containing a conversation interosseous (FDI) muscle over the primary motor cortex (M1). about job or school and a reading task were videotaped (Sony Active motor threshold (AMT) was determined as the Handycam DCR-TRV16E Mini DV digital Camcorder) and audio minimum intensity needed to evoke MEPs in the tonically recorded (Edirol R-09; sample rate: 16 bit/44.1 kHz; format: contracted contralateral FDI muscle of about 200 mV in five of WAV). SSI-3 norms were adapted from Riley (1994). Software ten consecutive trials. For the rTMS of the PMd the intersection for offline analysis were DivX player (DivX software, San of the coil was placed 2.5 cm anterior to the M1 representa- Diego) and WavePad (NCH software, Canberra). The offline tional hot spot of FDI. This procedure is in accordance with analysis of dysfluencies included 500 syllables for the previous studies (Doumas et al., 2005; Mochizuki et al., 2004; conversation and not less than 340 syllables for the reading Pollok et al., 2008; Schluter et al., 1998) and fits with func- task. Sound prolongations, blocks (silent prolongation of an tional imaging data displaying the PMd to be positioned about articulatory posture), sound and syllable repetitions were 1.8e2.5 cm (Picard and Strick, 2001) and 2.0 cm (Fink et al., counted as stuttered syllables. Monosyllabic words that were 1997) anterior to the M1 hand area. The coil was held with repeated with apparent undue stress or tension were counted the handle pointing backward and rotated away from the too (Sandrieser and Schneider, 2008). Furthermore, the esti- midline by 45 to induce a final anterioreposterior directed mated duration of the three longest blocks and observation of current in the stimulated cortex. Surface electromyogram physical concomitants were included for the estimate of (EMG) was recorded from the FDI through a pair of silveresilver stuttering severity in AWS. chloride surface electrodes in a belly-tendon montage. Raw signals were amplified, band-pass filtered (2e2500 Hz), digi- 2.3. Procedure tized with a micro 1401 AD converter (Cambridge Electronic Design, Cambridge, United Kingdom) and controlled by Signal The experiment consisted of two sessions, one for stimulating Software (Cambridge Electronic Design, version 2.13). the left and the other for stimulating the right PMd. During Complete muscle relaxation was controlled through visual each session participants performed one run of left index and feedback of EMG activity. Subthreshold rTMS was applied at one run of right index finger tapping before rTMS. Both runs 90% of ipsilateral AMT intensity for 20 min at 1 Hz over the left were repeated immediately (about 30 sec) after rTMS. The PMd in one session and the right PMd in another. This rTMS order of stimulation site and hand was counterbalanced protocol has been shown to decrease cortico-spinal excit- across participants. To avoid carry-over effects of the ability for several minutes (Gerschlager et al., 2001; Walsh and magnetic stimulation the second rTMS session was performed Rushworth, 1999) and complies with safety recommendations not less than 48 h after the first one. (Rossi et al., 2009; Wassermann, 1998). Participants sat in a silent room in front of a computer keyboard connected to the computer via a PS/2 cable. The 2.5. Data analysis keyboard was shielded to the participant’s visual field. Partic- ipants were requested to synchronize their unimanual index The mean values of the two dependent variables, asynchrony finger taps with a metronome. The acoustically presented and ITI-variability, were calculated separately for each group metronome signals contained clicks of 10 msec duration with (AWNS/AWS), each hand (left hand/right hand) and each site an inter click interval of 800 msec. Each experimental run of stimulation (left rTMS/right rTMS); thus, yielding 16 values comprised a continuous series of 56 clicks. The clicks were of asynchrony and ITI-variability, respectively.

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To control for group differences in the finger tapping calculated with a repeated measures ANOVA with hand as performance before rTMS we compared the individual mean a within-subjects factor and group as a between-subjects factor. baseline asynchrony values using a two-way mixed design Statistics were performed by SPSS Statistics 17.0 (http:// analysis of variance (ANOVA) with the between-subjects www.spss.com/de/software). factor group (AWS/AWNS) and the within-subjects factor hand (left/right). A similar ANOVA was calculated with the baseline ITI-variability. 3. Results At baseline finger taps preceded the acoustic signal in most participants resulting in negative asynchrony values. At baseline the two-way mixed design ANOVA with asyn- However, there were two AWS and seven AWNS that showed chrony values before rTMS as dependent variable revealed no a positive asynchrony in most runs. To test the impact of rTMS significant difference between AWS and AWNS (factor group ¼ ¼ we therefore normalized the asynchrony after stimulation for F1,27 1.4; p .3). However, the ANOVA revealed a more each participant and each session by subtracting the asyn- pronounced negative asynchrony in the right hand than in the ¼ ¼ chrony before stimulation. left hand [factor hand F1,27 7.73, p .01; left hand We entered the normalized values in a three-way mixed 28 52 msec (mean SD) vs right hand 39 60 msec]. design ANOVA with the between-subjects factor group (AWS/ Analysis yielded no further effect. ITI-variability before rTMS AWNS) and the within-subjects factors stimulation site (rTMS revealed no main effect or interaction for group and hand. over the left PMd/rTMS over the right PMd) and hand (left/ After rTMS, the analysis of normalized asynchrony values ¼ ¼ right). In addition, the expected rTMS-induced increases of revealed no main effects of hand (F1,27 1.5, p .2), stimulation ¼ ¼ ¼ ¼ asynchrony values (Pollok et al., 2008) were tested with one- site (F1,27 .4, p .5)andgroup(F1,27 .6, p .4) but a significant ¼ tailed t-tests. We tested the impact of rTMS on ITI-variability interaction between hand, stimulation site and group (F1,27 5.82, similarly by entering the normalized to baseline values. p ¼ .023). Post-hoc one-tailed t-tests, not corrected for multiple To exclude differences of age between groups we used comparisons (Perneger, 1998), revealed that rTMS over the left a two-tailed t-test for independent samples, for education, PMd significantly increased left hand asynchrony in AWNS handedness and percentage of stuttered syllables, we used (T ¼ 1.9, p ¼ .036) as previously shown (Pollok et al., 2008). By ManneWhitney U-tests. Nonparametric testing was chosen contrast, in AWS rTMS over the right PMd resulted in a significant since education is an ordinally scaled variable and handedness increase of left hand asynchrony (T ¼ 2.34, p ¼ .015) (Fig. 1). as well as percentage of stuttered syllables did not show After rTMS the analysis of normalized ITI-variability normal distribution in AWNS. The AMT comparison was revealed no main effects of group, stimulation site or hand

Fig. 1 e Mean values (±standard error) of normalized asynchrony (upper graphs) and change of normalized ITI-variability in percent with respect to baseline (lower graphs) after rTMS over the left and right PMd in AWS and AWNS. The analysis of asynchrony values yielded a three-way interaction between group (AWS/AWNS ), hand (left/right) and localization of rTMS (left PMd/right PMd ). Repetitive TMS over the left hemisphere prolonged left hand asynchrony in AWNS, but not in AWS. By contrast, right rTMS prolonged left hand asynchrony in AWS, but not in AWNS. ITI-variability increased after rTMS over the left PMd in AWNS. There was no signiﬁcant rTMS effect on ITI variability in AWS. Asterisks indicate p < .05.

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and no interactions of any of these factors. Interaction behavioral (Curry and Gregory, 1969; Sommers et al., 1975), between group, hand and stimulation site was marginally physiological (Biermann-Ruben et al., 2005; Moore and Lang, ¼ ¼ significant (F1,27 5.82, p .06). Post-hoc one-tailed t-tests, not 1977) and neuroimaging studies (Braun et al., 1997; De Nil corrected for multiple comparisons, yielded in an increased and Brutten, 1991; Ingham et al., 2004; Preibisch et al., 2003) normalized ITI-variability after rTMS over the left PMd in the provide evidence for a cerebral imbalance in AWS with an left hand of AWNS (T ¼2.01, p ¼ .032). This is in concordance increased involvement of the right hemisphere during speech with previous findings (Pollok et al., 2008). All other statistics production. Our results are in line with neural imaging studies yielded no significant differences (Fig. 1). suggesting an aberrant role of the left PMd (Lu et al., 2010a) and an additional involvement of the right PMd during speech (Braun et al., 1997; Fox et al., 1996; Ingham, 2001) and even 4. Discussion non-speech tasks in AWS (Chang et al., 2009; Morgan et al., 2008). Accordingly, using functional magnetic resonance We studied the cortical control of auditory paced finger move- imaging right hand finger tapping has been shown to be ments in AWS and AWNS. In AWNS, rTMS over the left PMd associated with bilateral pre- and post-central activation with increased left hand asynchrony and increased ITI-variability, increased activation of the right hemisphere in AWS as whereas rTMS over the right PMd was ineffective. By contrast, compared to AWNS (Morgan et al., 2008). Thus, less activation in AWS rTMS over the left PMd was ineffective, whereas rTMS of the left premotor area and stronger activation of the right over the right PMd prolonged left hand asynchrony. premotor area are not specific for speech in AWS, an interpretation corroborated by the present findings. 4.1. Left-hemispheric dominance on movement timing control in AWNS 4.3. No effects on right hand performance in both groups

In AWNS rTMS over the left PMd increased asynchrony and Previous studies documented contradictory data resulting ITI-variability of the left hand. This finding agrees well with from rTMS over the left PMd on right hand movement timing previous studies confirming a particular role of the left PMd in in non-stuttering adults (Del Olmo et al., 2007; Doumas et al., auditory paced rhythmic finger tapping (Pollok et al., 2009, 2005; Pollok et al., 2008). The present study showed an effect of 2008). Although it is not entirely clear via which connections left PMd rTMS on the subdominant left hand only. Within our the left PMd exerts dominance over the right hemisphere, sample of fluently speaking participants right handedness a specific significance of direct left PMd e right M1 connec- was less strongly developed (group average 76; median 70). tions (Pollok et al., 2008; Boroojerdi et al., 1996; Ferbert et al., Thus, one might speculate that the rTMS effect occurs in 1992) as well as subcortical circuits (Chouinard et al., 2003) strongly developed right handedness only (i.e., Edinburgh has been evidenced. It is well established that the cerebellum Inventory score of 90e100). To insure that the degree of is closely connected to the cerebral cortex via a cerebello- handedness did not interfere with our main result we recal- thalamo-cortical loop (Horne and Butler, 1995) and that audi- culated our statistics with three-way mixed analyses of tory paced isochronous tapping engages the cerebellum (Ivry covariance (ANCOVAs) with handedness scores as additional et al., 2002; Spencer et al., 2005). Even perception of an covariate. The ANCOVAs confirmed the three-way interaction ¼ isochronous rhythm involves the left PMd in concert with the between hand, site and group for asynchrony (F1,26 6.28, right cerebellum in healthy subjects suggesting the engage- p ¼ .019) and the marginal interaction of the same factors for ¼ ¼ ment of prediction mechanisms that are used for motor ITI-variability (F1,26 3.58, p .07). ANCOVAs yielded no preparation (Bengtsson et al., 2009). Therefore, the left PMd further effects. might serve as an interface between sensory prediction and Hence, the lack of modulation of right hand asynchrony temporally precise motor initiation (Kurata et al., 2000; cannot be explained by less pronounced right handedness Ramnani and Passingham, 2001). Consequently, an rTMS- within the present sample. Our data suggest that networks induced dysfunction of the left PMd might alter the functional controlling the performance of the non-dominant hand may connectivity of the cerebello-thalamo-cortical loop which be more susceptible to rTMS effects than those controlling the results in less precise timed motor behavior. dominant hand (Meyer-Lindenberg et al., 2002). This idea is This finding is consistent with a hemispheric dominance of also supported by a former diffusion tensor imaging study the left PMd in AWNS reported by Koch et al. (2006) during showing decreased fractional anisotropy underneath the pre- a response selection task and by Pollok et al. (2008) for central gyrus of the non-dominant hand related to the domi- movement timing during auditory paced finger tapping. nant hand (Buchel et al., 2004). Thus, morphologically the Nevertheless, this hypotheses is not unchallenged since in non-dominant hand relies on white matter with less integrity a response selection experiment, O’Shea et al. (2007) did not contrasted to the dominant hand. Furthermore, rTMS studies find evidence for such a hemispheric dominance. Rather, they demonstrate an improvement of non-dominant left hand demonstrated that changes in functional connectivity occur performance after inhibition of the ipsilateral left M1 in the pathway linking PMd and contralateral M1. (Kobayashi et al., 2004), but no improvement of dominant right hand performance after inhibition of the ipsilateral right 4.2. Right-shifted control of movement timing in AWS M1 (Weiler et al., 2008). Additionally, in AWNS, interhemispheric inhibition from the dominant to the non-dominant M1 In contrast to AWNS, right rTMS prolonged left hand asyn- is stronger than vice versa (Netz et al., 1995; Samii et al., 1997). chrony in AWS, whereas left rTMS was ineffective. Previous These results are compatible with our hypothesis that the

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network subserving motor control of the dominant hand populations like the right PMd that are additionally recruited might be more stable and thus, less prone to disturbance. for functional reorganization.

4.4. Why did right PMd stimulation affect the 4.5. Limitations of the study contralateral hand in AWS, while left PMd stimulation did affect the ipsilateral hand in AWNS? Although we used a standard procedure for determining rTMS location, we did not verify the exact PMd localization In both groups rTMS affected the subdominant hand. but, in by structural or functional imaging. We therefore cannot AWNS this effect occurred after left PMd stimulation, whereas rule out an aberrant structural or functional organization of in AWS right PMd stimulation yielded reduced timing accu- the left PMd in AWS. Cerebellar regions play an important racy. Although speculative, this result supports the hypoth- role for event timing (Spencer et al., 2003)andaltered esis that in AWS motor functions are shifted to the right auditory feedback (Howell and Sackin, 2002; Tourville et al., hemisphere. Thus, rTMS of the dominant hemisphere might 2008), and behavioral evidence indicated cerebellar deficits affect temporal accuracy of the subdominant hand. in children who stutter (Howell et al., 1997, 2008). However, Interestingly, the present data did not indicate differences we did not stimulate the cerebellum, because this proce- between AWS and AWNS prior to rTMS. Nevertheless, even dure is quite uncomfortable and may induce changes of the a task which is not impaired in AWS, like unimanual auditory cortico-spinal excitability. This effect is related to the paced finger tapping (Hulstijn et al., 1992; Max and Yudman, peripheral stimulation of the neck muscles rather than the 2003; Melvine et al., 1995; Zelaznik et al., 1994), is associated stimulation of the cerebellum itself (Gerschlager et al., with altered brain functions. Since in our study, inhibition of 2002). the right PMd elicited an aggravation of asynchrony but inhibition of the left PMd did not elicit an effect, we assume that the right PMd involvement reflects a compensatory 5. Conclusion mechanism rather than malfunction (Braun et al., 1997; Fox et al., 2000; Ludlow, 2000; Preibisch et al., 2003). The present findings indicate a right-shifted neuronal orga- This compensatory mechanism might be needed because nization for movement timing in AWS supporting the in AWS a basal neural deficit has been described in a left hypothesis of a generally altered neurophysiological organi- frontal brain region near the stimulation site. White matter zation of the motor control system in AWS. Since synchro- integrity is reduced in the left Rolandic Operculum in adults nization accuracy prior to rTMS did not differ between AWS (Sommer et al., 2002; Watkins et al., 2008) and adolescents and AWNS we suggest that the increased involvement of the who stutter (Chang et al., 2008). This results in a disconnection right PMd in non-speech and possibly also in speech tasks within the cerebral network processing speech motor represents a compensatory rather than a maladaptive behavior. Evidence in favor of such a weakened connection process. has been given by an abnormal activating time course of left premotor and primary motor regions (Salmelin et al., 2000) and altered left frontal-right cerebellar interactions (Lu et al., Funding 2010a)inAWS. The integration of motor areas of an undamaged hemi- This work was supported by the Deutsche sphere to adaptively compensate for damaged or discon- Forschungsgemeinschaft [SO 429/2-2 to M.S.]; Scholarship of nected regions has been recently identified in recovered the Stifterverband fu¨ r die Deutsche Wissenschaft, Walter und stroke patients (Johansen-Berg et al., 2002; Riecker et al., 2010). Ilse Rose Stiftung [to W.P.]; the Bernstein Center for Compu- Interestingly, a functional connectivity analysis pinpointed tational Neuroscience (BCCN) [01GQ0432, 01GQ0432 to W.P.] the SMAs to provide a driver-like input to the contralesional and the Forschungskommission of the Medical Faculty of the premotor and sensorimotor cortices in stroke patients Heinrich-Heine University Duesseldorf [9772328 to B.P.]. (Riecker et al., 2010). Nicole Neef, Kristina Jung, Holger Rothkegel, Dr. Bettina Pol- Anterior parts of the SMA are mainly connected with M1, lok, Dr. Alexander Wolff von Gudenberg, Prof. Dr. Walter PM and the putamen, posterior parts are mainly connected Paulus and Dr. Martin Sommer reported no biomedical with the inferior frontal gyrus, medial parietal, superior financial interests or potential conflicts of interest. frontal cortex and the caudatum (Johansen-Berg et al., 2004; Kim et al., 2010; Lehericy et al., 2004). In AWS, SMA shows increased activation during speech production (Chang et al., 2009) and even a more pronounced activation during stut- Acknowledgements tered as compared to fluent speech production (Ingham et al., 2000), which is also mirrored in a correlation between stutter- We are thankful to Manuel Hewitt for the technical assistance, rate and SMA activation (Fox et al., 2000). Additionally, the to Michael Nitsche for the support in statistical questions, to involvement of the putamen, which interacts with the SMA as Per Alm, Veronika Gutmann, Anne Smith, Vincent Walsh and well as with M1 and PM, is also altered in AWS (Braun et al., Christine Weber-Fox for fruitful discussions of the results, to 1997; Lu et al., 2010b; Ludlow and Loucks, 2003; Watkins Hayley Arnold for thoughtful comments and confirming et al., 2008). This over-activation may be related to the fact correct English usage and syntax, and to the reviewers for that the SMA supports the involvement of different neural their helpful feedback.

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Please cite this article in press as: Neef NE, et al., Right-shift for non-speech motor processing in adults who stutter, Cortex (2010), doi:10.1016/j.cortex.2010.06.007 Dopaminergic Potentiation of rTMS-Induced Motor Cortex Inhibition Nicolas Lang, Sascha Speck, Jochen Harms, Holger Rothkegel, Walter Paulus, and Martin Sommer Background: Experiments in animal models suggest that neuronal plasticity can be enhanced by dopaminergic receptor activation. The present study tested whether stimulation-induced plasticity of human motor cortex after low-frequency repetitive transcranial magnetic stimulation (rTMS) could be potentiated by a single oral dose of the combined D1/D2 receptor agonist pergolide. Methods: In a randomized, double-blind, placebo-controlled cross-over design, nine healthy young volunteers received .125 mg pergolide or placebo 2 hours before 1 Hz rTMS was applied for 20 min to the left primary motor cortex. In a control experiment 7 subjects received .125 mg pergolide 2 hours before sham rTMS. We used single-pulse TMS at rest to assess corticospinal excitability before and up to 24 min after rTMS. Results: Suppression of corticospinal excitability by 1 Hz rTMS was more pronounced after pergolide intake compared with placebo and lasted approximately 20 min after pergolide but only 5 min after placebo. No change of corticospinal excitability could be observed when sham rTMS was performed after pergolide intake. Conclusions: The results suggest a possible role for dopaminergic potentiation of rTMS-induced neuroplasticity in experimental or therapeutic applications and should be considered when rTMS is applied in patients under medication with dopamine agonists or antagonists.

Key Words: D1, D2, pergolide, primary motor cortex, receptor, the effects of plasticity as a consequence of D2 receptor activa- repetitive transcranial magnetic stimulation, stimulation-induced tion are less consistent: with regard to LTP it has been described plasticity as enhancing (19), suppressing (20), or without effect (16). Long-term depression has been shown to be enhanced by D2 receptor activation (21,22) but also to be inhibited (18). hanges in cortical excitability can be induced in humans non-invasively with repetitive transcranial magnetic stimula- The present study was designed to explore the effects of tion (rTMS) (1,2). Although the mechanisms of pergolide,these a combined D1/D2 receptor agonist, on motor cortex C excitability changes induced by low-frequency rTMS in a ran- changes are not fully understood, analogies to long-term potentiation (LTP) and long-term depression (LTD) of individual synapses domized, double-blind, placebo-controlled crossover design. are apparent. When rTMS is given with constant interpulse-intervals The preponderance of evidence suggests that pergolide, as a the direction of after-effects can be controlled by the frequency of D1/D2 agonist, would enhance LTD, although studies specifi- stimulation: lower frequencies, in the range of 1 Hz, can produce cally examining the impact of this drug on LTD are lacking. LTD-like inhibition of motor cortical excitability (3,4), whereas Ն frequencies of 5 Hz can induce LTP-like facilitation (5,6). Methods and Materials In recent years, rTMS has attracted considerable interest as a therapeutic tool in neuropsychiatry. The method has been used Altogether 14 healthy human subjects (8 women and 6 men, in numerous clinical trials to improve a variety of brain diseases, ages 21–44 years, median age 25 years) gave their informed such as Parkinson’s disease, epilepsy, major depression, and consent before participating in the experiments. Experimental schizophrenia (7,8). Because clinical effects have oftenprocedures been had the approval of the Ethics Committee of the subtle and variable, the therapeutic potential of rTMS is still open University of Goettingen and were performed according to the to debate, and methodological considerations to enhance rTMS ethical standards laid down in the Declaration of Helsinki. efficiency by optimizing stimulation pattern (9,10) or sensitizingIn the main experiment nine participants (six women and cortical areas with preconditioning (11–13) are developing.three men, ages 21–26 years, median age 24 years) underwent Another approach of potentiating rTMS effects might be two rTMS sessions on different days separated by at least 1 week. achieved by pharmacological interventions with dopaminergic One session was done after pergolide intake and one after drugs. There is evidence that dopaminergic mechanisms are placebo intake. A uniform capsule, containing .125 mg pergolide involved in N-methyl-D-aspartate (NMDA) receptor-dependent or placebo, was orally administered 2 hours before each exper- neuroplasticity. In animal models, LTP and LTD can be modified iment. The order of intake (pergolide or placebo) was pseudo- by D1 or D2 receptor activation: D1 receptor activity can have randomized and balanced, and subjects and TMS examiners enhancing effects on the induction and consolidation of LTP were not informed about it. At the end of each session subjects (14 –17) but can also facilitate LTD induction (17,18). wereReports interviewed on about possible adverse effects. The rTMS was given at a rate of 1 Hz over 20 min (i.e., 1200 pulses) with an From the Department of Neurology (NL), Christian-Albrechts University, intensity of 90% of the individual resting motor threshold (RMT) Kiel, Germany; and Department of Clinical Neurophysiology (SS, JH, HR, to the left primary motor cortex (M1). Corticospinal excitability WP, MS), Georg-August University, Goettingen, Germany. was studied over a period of 8 min before rTMS (baseline) and Address reprint requests to Dr. Nicolas Lang, Department of Clinical Neuro- again for 24 min after rTMS with single pulse TMS. Here TMS was physiology, Georg-August-University, Robert-Koch-Strasse 40, 37075 given to the left M1 at a rate of .25 Hz, and the intensity was Goettingen, Germany; E-mail: [email protected]. adjusted to yield baseline motor evoked potentials (MEP) in the Received November 21, 2006; revised April 11, 2007; accepted April 12, relaxed right first dorsal interosseus muscle (FDI) with mean 2007. peak-to-peak amplitudes of approximately 1 mV. This intensity

(SI1mV) was individually determined at the beginning of each experiment and held constant throughout the session. An additional control experiment was performed in seven subjects (four women and three men, ages 21–44 years, median age 24 years) with sham rTMS given 2 hours after a single oral dose of .125 mg pergolide in order to exclude an unspecific effect of pergolide intake on cortical excitability. For sham rTMS, the coil placed over the motor hot spot was disconnected from the stimulator during rTMS. The TMS stimulator was then dis- charged through a second coil, which was fixed to a coil holder positioned approximately 50 cm behind the subject’s head. This provides a similar noise compared with real rTMS and can serve as a reasonable method to provide sham rTMS (11). All other procedures were kept identical to the main experiment. TMS was performed with a Medtronic MagPro stimulator and a figure-of-eight–shaped Medtronic MC-B70 coil (Medtronic Func- Figure 1. Low-frequency repetitive transcranial magnetic stimulation tional Diagnostics, Skovlunde, Denmark). The coil was held tan- (rTMS)–induced motor cortex inhibition 2 hours after intake of .125 mg pergolide or placebo. Filled symbols indicate signiﬁcant differences of gentially to the skull over the optimal cortical representation of the mean motor evoked potential (MEP) amplitudes compared with before right FDI with the handle pointing posterolaterally at a 45-degree rTMS (post hoc t test; p Ͻ .05, error bars indicate SEM). angle to the sagittal plane. Stimuli were biphasic, and the first phase of the stimulus elicited an anterior–posterior current in the brain. effects for the factor time in both conditions [placebo: F(6,48) ϭ In each subject rTMS experiments were done at identical times 2.77, p ϭ .021; pergolide: F(6,48) ϭ 4.50, p ϭ .01]. Post hoc during the day with the participant comfortably seated in a reclining analyses showed that MEPs were more strongly suppressed after chair with head and arm rests. Surface electromyogram (EMG) was pergolide intake compared with the placebo condition within the recorded from the right FDI through a pair of silver–silver chloride first 20 min after rTMS (post hoc t tests; all p Ͻ .05). After placebo (Ag-AgCl) surface electrodes in a belly-tendon montage. Raw intake a significant inhibition compared with baseline values signals were amplified, band-pass filtered (3Hz–3kHz), digitized could only be seen within the first 4 min (1 time bin) after rTMS with a micro 1401 AD converter (Cambridge Electronic Design, (post hoc t test; p ϭ .039), whereas it lasted for 20 min (5 time Cambridge, United Kingdom) controlled by Signal Software (Cam- bins) after pergolide (post hoc t tests; all p Ͻ .05). At the last time bridge Electronic Design, version 2.13), and stored on a personal bin (21–24 min after rTMS) mean MEP values in both drug computer for offline analysis. Complete relaxation was controlled conditions were not different from baseline (Figure 1). through auditory and visual feedback of EMG activity. Mean values (Ϯ SEM) in the pergolide and placebo condition, Mean MEP values of each individual were calculated for baseline respectively, were: for baseline MEP .94 Ϯ .04 and .97 Ϯ .04 mV, recordings and for 6 time bins of 4-min duration each, covering a for RMT 41 Ϯ 2 and 41 Ϯ 2% maximum stimulator output, and for 24-min period after rTMS. For the main experiment these values Ϯ Ϯ SI1mV 50 2 and 49 2% maximum stimulator output. Analyses were entered into a two-way repeated-measures analysis of vari- of baseline MEP values, RMT, and SI1mV data did not reveal ance (ANOVA) with “drug” (two levels: pergolide and placebo) and significant differences between drug conditions for these data “time” (seven levels: before rTMS and time bins 1–6 after rTMS) as (t tests; all p Ͼ .5). within-subject factors. For the control experiment we performed a In the control experiment with sham rTMS after pergolide intake one-way ANOVA with “time” (seven levels: before rTMS and time no significant effect on “time” could be observed [F(6,36) ϭ 1.336, bins 1–6 after rTMS) as within-subject factor. Conditional on a p ϭ .267]. significant F value, we performed follow-up one-way ANOVAs and post hoc paired-samples two-tailed t tests to characterize the effects Discussion revealed by the main ANOVA. Two-tailed t tests were also used to The present study confirms previous studies (3,4,23) that continu- test for differences in RMT and SI1mV at baseline between drug Ͻ ous 1-Hz rTMS to the human motor cortex can induce a transient conditions. A p value of .05 was considered significant for all decrease in corticospinal excitability. Extending previous work, we statistical analyses. All results are given as mean and SEM. show that this effect can be potentiated by a single dose of the Results combined D1/D2 receptor agonist pergolide: suppression of corticospinal excitability was more pronounced after pergolide compared Three subjects reported some mild and transient adverse with placebo and lasted approximately 20 min after pergolide, whereas effects after the intake of pergolide, such as dizziness and it ceased within 5 min after placebo. No change of corticospinal nausea; however this did not interfere with the ability of the excitability could be observed when sham rTMS was performed after subjects to complete the study. None of the subjects reported pergolide intake. These findings are in line with results obtained from adverse effects after placebo intake. Side effects from pergolide animal experiments that show that dopamine receptor activation can might have partially unblinded the investigators and therefore enhance LTD (17,24). However, it should be considered that our data weakened the double-blinded study design. However, partici- has been obtained in motor cortex, and it cannot be assured that these pants did not make explicit statements to the investigators about conditions can be applied identically to other cortical areas. Moreover, adverse events until the end of each session. it has been shown that pergolide—at least on a peripheral level—not In the main experiment ANOVA on mean MEP revealed signif- only interacts with dopamine receptors but also with serotonin (5-HT) icant main effects of the factors “drug” [F(1,8) ϭ 11.31, p ϭ .01] and receptors and ion channels (25,26). Therefore, it cannot be ruled out “time” [F(6,48) ϭ 5.92, p ϭ .003] and a significant “time” ϫ “drug” completely that the observed central nervous system effects could interaction [F(6,48) ϭ 2.38, p ϭ .042]. Follow-up one-way ANOVAs partially be mediated by mechanisms other than dopaminergic recep- separately performed on each drug condition showed significant tor activation. www.sobp.org/journal N. Lang et al. BIOL PSYCHIATRY 2008;63:231–233 233

Our results parallel a recent study that tested effects of D1/D2 9. Huang YZ, Edwards MJ, Rounis E, Bhatia KP, Rothwell JC (2005): Theta receptor agonists and antagonists on excitability changes induced burst stimulation of the human motor cortex. Neuron 45:201–206. by transcranial direct current stimulation (tDCS) (27). Like10. ThickbroomrTMS, GW, Byrnes ML, Edwards DJ, Mastaglia FL (2006): Repetitive tDCS can be used to alter cortical excitability bi-directionally and paired-pulse TMS at I-wave periodicity markedly increases corticospinal excitability: A new technique for modulating synaptic plasticity. Clin non-invasively in humans (28), and its pharmaco-physiologicalNeurophysiol 117:61–66. properties suggest that activity-dependent synaptic plasticity, such 11. Lang N, Siebner HR, Ernst D, Nitsche MA, Paulus W, Lemon RN, et al. as LTP and LTD, mediates the effects induced by tDCS (2004):(29). PreconditioningWith with transcranial DC stimulation sensitizes the tDCS, combined D1/D2 receptor activation by pergolide also en- motor cortex to rapid-rate TMS and controls the direction of after- hanced LTD-like effects after inhibitory cathodal tDCS, whereas D2 effects. Biol Psychiatry 56:634–639. antagonism with sulpiride alone or D1 activation alone (achieved by 12. Siebner HR, Lang N, Rizzo V, Nitsche MA, Paulus W, Lemon RN, et al. (2004): combining pergolide and sulpiride) prevented inhibition. This was Preconditioning of low-frequency repetitive transcranial magnetic stimula- taken as an explanation that D2 receptor activation has a consoli- tion with transcranial direct current stimulation: evidence for homeostatic plasticity in the human motor cortex. J Neurosci 24:3379–3385. dation-enhancing effect on LTD-like cortical excitability changes. 13. Iyer MB, Schleper N, Wassermann EM (2003): Priming stimulation en- Although not tested in the present study, it might be questioned hances the depressant effect of low-frequency repetitive transcranial whether only LTD-like plasticity induced by rTMS could be potentiated magnetic stimulation. J Neurosci 23:10867–10872. by dopaminergic receptor activation or whether LTP-like plasticity, 14. BachME,BaradM,SonH,ZhuoM,LuYF,ShihR,etal.(1999):Age-relateddefects such as the facilitation of excitability by 5-Hz rTMS, would be similarly in spatial memory are correlated with defects in the late phase of hippocampal enhanced. In the aforementioned tDCS study no enhancement or long-term potentiation in vitro and are attenuated by drugs that enhance the prolongation of LTP-like effects after facilitatory anodal tDCS could be cAMP signaling pathway. Proc Natl Acad SciUSA96:5280–5285. observed with pergolide (27). However, with another non-invasive15. 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Could it be that dopaminergic receptor activation accompanyingfor the bidirectional modulation of synaptic plasticity in the prefrontal stimulation-induced plasticity can generally be used for “gating” of LTP- cortex by D1 receptors. Proc Natl Acad SciUSA101:3236–3241. and LTD-like processes in motor cortex? Further experiments will be 18. Chen Z, Ito K, Fujii S, Miura M, Furuse H, Sasaki H, et al. (1996): Roles of necessary to clarify this question. dopamine receptors in long-term depression: Enhancement via D1 receptors and inhibition via D2 receptors. Receptors Channels 4:1–8. In conclusion, the present study demonstrates that LTD-like 19. Manahan-Vaughan D, Kulla A (2003): Regulation of depotentiation and motor cortex inhibition induced by rTMS in humans can be en- long-term potentiation in the dentate gyrus of freely moving rats by hanced by a single dose of the D1/D2 receptor agonist pergolide. dopamine D2-like receptors. Cereb Cortex 13:123–135. 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www.sobp.org/journal Clinical Neurophysiology 117 (2006) 838–844 www.elsevier.com/locate/clinph

Half sine, monophasic and biphasic transcranial magnetic stimulation of the human motor cortex

Martin Sommer a, Ara´nzazu Alfaro a,b,c, Milena Rummel a, Sascha Speck a, Nicolas Lang a, Tobias Tings a, Walter Paulus a,*

a Department of Clinical Neurophysiology, University of Go¨ttingen, Robert-Koch-Street 40, 37075 Go¨ttingen, Germany b Instituto de Bioingenierıá, Unidad de Neuropro´tesis y Rehabilitacioń Visual, Carretera Alicante-Valencia, km 87, 03550 San Juan de Alicante, Spain c Departamento de Neurologıá, Hospital General Universitario, C/Pintor Baeza s/n, 03010 Alicante, Spain

See Editorial, pages 714–715

Abstract

Objective: To compare half sine transcranial magnetic stimuli (TMS) with conventional monophasic and biphasic stimuli, measuring resting and active motor threshold, motor evoked potential (MEP) input/output curve, MEP latency, and silent period duration. Methods: We stimulated the dominant hand representation of the motor cortex in 12 healthy subjects utilising two different MagPro stimulators to generate TMS pulses of distinct monophasic, half sine and biphasic shape with anteriorly or posteriorly directed current flow. Results: The markedly asymmetric monophasic pulse with a posterior current flow in the brain yielded a higher motor threshold, a less steep MEP input/output curve and a longer latency than all other TMS types. Similar but less pronounced results were obtained with a less asymmetric half sine pulses. The biphasic stimuli yielded the lowest motor threshold and a short latency, particularly with the posterior current direction. Conclusions: The more asymmetric the monophasic pulse, the stronger the difference to biphasic pulses. The 3rd and 4th quarter cycle of the biphasic waveform make it longer than any other waveform studied here and likely contribute to lowering motor threshold, shortening MEP latency and reversing the influence of current direction. Significance: This systematic comparison of 3 waveforms and two current directions allows a better understanding of the mechanisms of TMS. q 2006 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.

Keywords: Transcranial magnetic stimulation; Stimulus conﬁguration; Monophasic; Biphasic; Motor cortex

1. Introduction The half sine pulse provided by the MagPro X100 MagOption stimulator (Medtronic Inc., Minneapolis, MN, The difference between biphasic and monophasic USA) is identical to the biphasic pulse of that machine, but transcranial magnetic stimulation (TMS) is increasingly cut after the second quarter cycle. For the monophasic noticed (Corthout et al., 2001; Kammer et al., 2001; Niehaus waveform, the second quarter cycle is cut earlier and then et al., 2000) and taken into account also in the ﬁeld of gradually tapers off and therefore differs markedly from the repetitive TMS (rTMS) (Arai et al., 2005; Sommer et al., second quarter cycle of the biphasic pulse. For comparison, 2002; Tings et al., 2005). The exact reason for differences, note that the Magstim 200 stimulator (The Magstim e.g. with regard to motor threshold is still debated, so is the Company, Whitland, Dyfed, UK) generates a monophasic role of the different components of a biphasic pulse in vivo pulse, and the Magstim Rapid and Rapid2 stimulator and in vitro (Kammer et al., 2001; Maccabee et al., 1998). generate biphasic pulses (Fig. 1). We were curious to learn whether the half sine * Corresponding author. Tel.: C49 551 39 66 50; fax: C49 551 39 81 26. waveform, being in between the monophasic and the E-mail address: [email protected] (W. Paulus). biphasic waveform, gives any insight about the role of the

1388-2457/$30.00 q 2006 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.clinph.2005.10.029 M. Sommer et al. / Clinical Neurophysiology 117 (2006) 838–844 839

Fig. 1. Current induced in a probe coil of 1 cm diameter by different types of transcranial magnetic stimulators, recorded and stored by an oscilloscope (LeCroy 9350C, Chestnut Ridge, NY, USA). Stimulus intensity as annotated in percent of maximum stimulator output. Upper part, waveforms studied in this manuscript. Upper left, waveform induced by the old (green) MagPro stimulator in the ‘monophasic’ mode. Upper middle and upper right, waveforms induced by the MagPro X100 MagOption in the ‘half sine’ mode (upper middle) and in the ‘biphasic’ mode (upper right). For all upper graphs, the same Dantec MC- B70 coil was used. Lower part, other waveforms induced by different Magstim stimulators recorded with the same set-up and illustrated for comparison. Identical ENG SP2 8606 ﬁgure-8-coil for Magstim 200 and Magstim Rapid; P/N 3110 00 S/N074 ﬁgure-8-coil for Magstim Rapid2.

TMS waveform with regard to typical measures of Go¨ttingen. For all experiments we used the same slightly corticospinal excitability such as motor threshold, motor bent figure-of-8 coil (MC B70, Dantec S.A., Skovlunde, evoked potential (MEP) latency, MEP input/output curve Denmark) either with the old, green MagPro stimulator (old and silent period duration, and therefore compared all 3 version, produced earlier than 2003) or a new, white MagPro types of pulses in the same group of subjects. We could not X 100 MagOption stimulator (produced from 2003 on). study other measures such as short-interval (Kujirai et al., 1993; Ziemann et al., 1996) or long-interval intracortical 2.1. Six types of TMS inhibition (Chen, 2004) because of technical limitations to the monophasic pulse repetition rate. During the preparation of this project it turned out that the MagPro X100 MagOption stimulator used in our lab was not able to produce a truly monophasic pulse, but produced 2. Material and methods a half sine pulse instead even when waveform switch was set at monophasic. Therefore, we had to use the monophasic All measures were studied in 12 healthy subjects (mean pulse from the old MagPro stimulator (Dantec S.A., age 27.5, range 22–35 years, 6 women) with no previous or Skovlunde, Denmark). current medical or neurological disease and no intake of During the review process of this manuscript, one medication except for oral contraceptives in two of the 6 anonymous reviewer indicated that the MagPro X100 women. In all subjects we studied the dominant hand, MagOption used in his/her laboratory is able to generate stimulating the contralateral motor cortex. Eleven of 12 all 4 types of waveforms correctly (monophasic, half sine, subjects were right-handed with a mean Olfield 10-items biphasic, biphasic burst). We therefore had the machine handedness score of 87.9G18.6 of 100 points of right- used in our lab tested by the manufacturer, who replaced a handedness (meanGSD) (Oldfield, 1971). The experiments faulty monophasic clamp diode. After repair, the MagPro were approved by the ethics committee of the University of X100 MagOption we are using is able to generate all 4 types 840 M. Sommer et al. / Clinical Neurophysiology 117 (2006) 838–844 of waveforms correctly. However, for the data presented Welwyn Garden City, UK). For analysis we used a here and generated before the repair, we had to use the old repeated-measures ANOVAs with intensity (3 levels), MagPro to generate truly monophasic pulses. waveform (3 levels) and current direction (two levels) as To investigate 6 types of TMS (old MagPro monophasic within-subjects factors. Post-hoc t tests were paired and anteriorly directed stimuli, old MagPro monophasic poster- two-tailed. Similar follow-up ANOVAS were calculated iorly directed stimuli, half sine anteriorly and posteriorly separately comparing monophasic and half sine as well as directed stimuli from the MagPro X100 MagOption half sine and biphasic pulses. stimulator, and biphasic anteriorly and posteriorly directed In addition, we measured the mean MEP onset latency stimuli from the MagPro X100 MagOption stimulator), the from the 10 trials with RMT C20 A/ms intensity in each subjects were studied in 3 separate sessions using two kind subject and calculated a factorial ANOVA with pulse type of pulse waveforms in each one. The order of pulse (6 levels) as between-subjects factor. Post-hoc t tests were waveforms was randomized for each volunteer, and subjects paired and two-tailed. Similar follow-up ANOVAS were were not told any details about the TMS types. Experiments calculated separately comparing monophasic and half sine took place at least half an hour apart from each other. In this as well as half sine and biphasic pulses. manuscript the current direction is always indicated as the initial current flow in the brain, which is opposite to the 2.4. Silent period current direction in the coil (Bohning, 2000). We recorded the contralateral silent period (cSP) while 2.2. Motor threshold the subjects abducted the small finger of the investigated hand at about 20% maximum voluntary contraction, In each experimental session, we first determined the monitored by acoustic and visual feedback (Myograph II, optimal dominant abductor digiti minimi (ADM) motor Toennies GmbH, Freiburg, Germany). We used 10 pulses representation, since it is not necessarily identical for all each at RMT C10 A/ms, RMT C20 A/msandRMT types of TMS, and marked the respective spot with a pen on C25 A/ms. In each trial the cSP duration was measured the skin. We determined the resting motor threshold (RMT), off-line from the onset of the MEP to the reoccurrence of i.e. the lowest intensity that yielded motor evoked potentials any sustained EMG activity (Orth and Rothwell, 2004). For (MEPs) of O50 mV from the muscle at rest in at least 5 of 10 analysis we calculated a repeated-measures ANOVA with consecutive trials, and the active resting threshold (AMT), intensity (3 levels), waveform (3 levels) and current i.e. the lowest intensity that yielded motor evoked potentials direction (two levels) as within-subjects factors. Post-hoc t of O250 mV from the tonically contracted muscle in at least tests were paired and two-tailed and calculated on the 5 out of 10 consecutive trials (Rothwell et al., 1999). pooled values of all intensity levels. Similar follow-up Step width for threshold determination was about 1% of ANOVAS were calculated separately comparing mono- stimulator output. For ADM electromyography we used phasic and half sine as well as half sine and biphasic pulses. silver–silverchloride electrodes in a belly-tendon montage. For recording we used the ‘Signal’ software and a CED 1401 hardware (Cambridge Electronic Design, Cambridge, 3. Results UK) at a sampling rate of 10,000 Hz and filtered at 1.6 Hz and 1 kHz. For analysis we calculated a repeated-measures 3.1. Side effects ANOVA with threshold (RMT, AMT), waveform (mono, half sine, bi) and current direction (anterior, posterior) as No side effects of TMS were reported. within-subjects factors. Post-hoc t tests were paired and two-tailed. Similar follow-up ANOVAS were calculated 3.2. Motor threshold separately comparing monophasic and half sine as well as half sine and biphasic pulses. The motor threshold was differentiated according to the type of stimulation and the current direction (Fig. 2). It was 2.3. MEP I–O curve; MEP latency generally higher with monophasic than with half sine or biphasic pulses. For monophasic and half sine stimulation, For studying the MEP I–O curve, we applied 10 stimuli the posteriorly oriented pulses yielded a higher motor each for the intensity of RMT, RMT C10 A/ms, and RMT threshold than the anteriorly oriented currents, the differ- C20 A/ms. We did not use intensity steps relative to the ence being most pronounced for the monophasic configur- threshold, since this would have resulted in unequal step ation. By contrast, for biphasic stimulation posteriorly widths for the different types of TMS. We normalized the oriented stimuli yielded lower thresholds than anteriorly raw values to the maximal M-wave amplitude determined oriented ones (repeated-measures ANOVA, effect of by supramaximal peripheral stimulation using a conven- threshold (RMT vs. AMT), F(1,11)Z104.5, P!0.0001; tional EMG stimulation block and an electrical stimulator effect of waveform, F(2,22)Z107.8, P!0.0001; no effect (Multipulse Stimulator model D 185, Digitimer Inc., of current direction; interaction of threshold by waveform, M. Sommer et al. / Clinical Neurophysiology 117 (2006) 838–844 841

100 27

90 A-P 26.5 A-P 80 P-A 26 * P-A 70 25.5 * 60 * 25 50 * * 24.5 40 24 30 MEP latency [ms]

Stimulus intensity [A/µs] 20 23.5 10 23

0 22.5 Mono Half Bi Mono Half Bi Mono Half Bi sine sine sine RMT AMT Fig. 3. Motor evoked potential latency (ms), meanGSE. Asterisks indicate Fig. 2. Motor threshold with the target muscle at rest (RMT) or during significant post-hoc differences between current directions for a particular voluntary target muscle contraction (AMT), shown for the 6 types of TMS waveform. studied, meanGSE. Asterisks indicate significant post-hoc differences between current directions for a particular waveform. effect of current direction, F(1,11)Z30.7, P!0.0001; interaction of waveform by current direction, F(1,11)Z Z Z F(2,22) 7.3, P 0.0037; interaction of waveform by 10.2, PZ0.009), but not between half sine and biphasic current direction, F(2,22)Z52.6, P!0.0001). Post-hoc t pulses (repeated-measures ANOVA, effect of waveform, tests yielded a significant difference between all waveforms. F(1,11)Z4.4, PZ0.059, effect of current direction, PZ Follow-up ANOVAs yielded a difference between 0.95; no interaction). monophasic and half sine pulses (repeated-measures ANOVA, effect of threshold, F(1,11)Z118.6, P!0.0001; 3.4. MEP I–O curve effect of waveform, F(1,11)Z23.9, PZ0.0005, effect of current direction, F(1,11)Z17.3, PZ0.002; interaction of The increase of MEP amplitude with rising stimulus waveform by current direction, F(1,11)Z23.1, PZ0.0005; intensity was least steep with the monophasic posteriorly interaction of threshold by waveform, F(1,11)Z4.8, PZ oriented pulses (repeated-measures ANOVA, effect of 0.052) as well as between half sine and biphasic pulses intensity, F(2,22)Z35.6, P!0.0001; no main effect of (repeated-measures ANOVA, effect of threshold, F(1,11)Z waveform or current direction; interaction of intensity by 129.8, P!0.0001; effect of waveform, F(1,11)Z90.9, P! waveform, F(4,44)Z3.67, PZ0.011; Fig. 4). Post-hoc tests 0.0001, effect of current direction, PZ0.18; interaction of yielded a significant difference between the posteriorly waveform by current direction, F(1,11)Z37.9, P!0.0001, oriented currents of the monophasic type and the half sine as no other interaction). well as the biphasic pulse. Follow-up ANOVAs yielded a difference between 3.3. MEP latency monophasic and half sine pulses (repeated-measures ANOVA, effect of intensity, F(2,22)Z30.2, P!0.0001; The MEP latency was significantly different between effect of waveform, F(1,11)Z3.9, PZ0.071, effect of waveforms. The pattern resembled that of the motor threshold (Fig. 3), with longer latencies for the posteriorly oriented pulses, particularly in the case of monophasic 22.5 pulses, and again a reversed situation for the biphasic 20.0 17.5 waveform where anteriorly oriented pulses yielded slightly Biphasic, A-P 15.0 longer latencies (repeated-measures ANOVA, effect of Mono, A-P waveform, F(2,22)Z19.3, P!0.0001; effect of current 12.5 Half sine,A-P direction, F(1,11)Z14.9, PZ0.003; interaction of wave- 10.0 Biphasic, P-A Z ! 7.5 Mono, P-A form by current direction, F(2,22) 15.1, P 0.0001). Post- to M-wave [%] hoc t tests yielded a significant difference between 5.0 * Half sine,P-A monophasic and half sine waveforms as well as between MEP amplitude normalized 2.5 the monophasic and the biphasic waveforms. 0 * Follow-up ANOVAs yielded a difference between RMT RMT + 10 A/µs RMT + 20 A/µs monophasic and half sine stimuli (repeated-measures Fig. 4. MEP amplitude input/output curves tested with fixed step widths; ANOVA, effect of waveform, F(1,11)Z23.0, PZ0.0006, meanGSE. Asterisks indicate significant differences in post-hoc t tests. 842 M. Sommer et al. / Clinical Neurophysiology 117 (2006) 838–844 current direction, F(1,11)Z5.0, PZ0.046; interaction of 4. Discussion intensity by waveform, F(2,22)Z2.7, PZ0.09; interaction of intensity by current direction, F(2,22)Z3.6, PZ0.044, This study expands earlier data (Kammer et al., 2001; no other interaction), but not between half sine and biphasic Niehaus et al., 2000) by showing that when probing pulses (repeated-measures ANOVA, effect of intensity, measures of corticospinal excitability with single pulse F(2,22)Z40.8, P!0.0001; no effect of waveform, PZ0.7; TMS the half sine pulses of the new MagPro TMS no effect of current direction, no significant interaction). stimulator yield results in between those of the monophasic pulses and the biphasic configuration. This holds true for motor threshold, MEP latency and SP duration. Our data 3.5. Silent period also show that the influence of the current direction is of the same type for the monophasic and the half sine pulse, The contralateral silent period duration was shortest with though more pronounced for the first than the latter, but the monophasic pulses, intermediate with half sine pulses, reversed for the biphasic waveform. and longest with the biphasic pulses. For monophasic and The MEP latency difference is consistent with findings of half sine pulses the posteriorly oriented currents yielded a earlier reports (Day et al., 1989; Di Lazzaro et al., 2001; slightly longer SP duration than the anteriorly oriented Sakai et al., 1997). It has been suggested that either different pulses, the reverse was true for the biphasic pulses (Fig. 5). structures might be activated by different waveforms and A repeated-measures ANOVA yielded an effect of intensity, current directions, or that the same sets of interneurons in F(2,22)Z59.7, P!0.0001; an effect of waveform, the motor cortex are activated at different sites (Di Lazzaro F(2,22)Z14.6, P!0.0001; no effect of current direction, et al., 2001; Kammer et al., 2001). This assumption was and no significant interaction. Post-hoc tests yielded a based on epidural recordings of corticospinal volleys. Using significant difference between all waveforms and between this technique, Di Lazzaro and colleagues described a all levels of intensity. preferential activation of the I1-wave by anteriorly oriented Follow-up ANOVAs yielded a difference between pulses at low threshold, whereas posteriorly oriented pulses monophasic and half sine pulses (repeated-measures tended to elicit later I-waves with a higher threshold. The ANOVA, effect of intensity, F(2,22)Z63.9, P!0.0001; order of I-wave recruitment and the relative latencies of the effect of waveform, F(1,11)Z8.7, PZ0.013, effect of I-wave peaks were however not in all subjects exactly current direction, PZ0.34; no significant interaction), and reversed with the opposite orientation, and the correlation in particular between half sine and biphasic pulses between I-wave-amplitudes and MEP amplitude was not the (repeated-measures ANOVA, effect of intensity, same for either direction (Di Lazzaro et al., 2001), raising F(2,22)Z42.2, P!0.0001; effect of waveform, F(1,11)Z questions about the exact site of stimulation. 5.8, PZ0.035, effect of current direction, PZ0.89; With regard to the motor threshold, it should be kept in interaction of intensity by waveform, F(2,22)Z6.4, mind that the stimulus intensity indicated by the hardware is w PZ0.006, interaction of intensity by current direction, only derived from the peak current in the initial 50 msof F(2,22)Z2.7, PZ0.087; interaction of waveform by current the pulse (Mr Kienle, Medtronic, personal communication). direction, F(1,11)Z4.3, PZ0.061; no other interaction). It does therefore not predict the physiological relevance of the latter parts of the damped oscillation, which may be different for the various waveforms even if the initial peak current is identical. Hence, it is important to indicate both 190 A-P intensity and waveform to precisely designate a given pulse. 185 P-A In our study, the MEP input/output curves from 180 monophasic posteriorly oriented pulse differed most * 175 strongly from the other types. It is tempting to speculate that the relatively strong stimuli required with that TMS 170 condition (co)activate sets of interneurons that are different 165 or more numerous than for the other types of TMS. 160 Furthermore, the monophasic pulse is the most asymmetric 155 one and there fore the fast initial current flow is least

Silent Period duration [ms] 150 counterbalanced by the opposite current flow of the second 145 quarter cycle. 140 Maccabee and colleagues hypothesized that the physio- Mono Half Bi logically relevant part of the biphasic waveform is the second sine quarter cycle oriented oppositely to the initial quarter cycle Fig. 5. Contralateral silent period duration with 3 levels of stimulus (Maccabee et al., 1998). This was confirmed in vivo by intensities pooled; meanGSE. Asterisks indicate significant post-hoc Corthout and colleagues with regard to skotoma induction by differences between current directions for a particular waveform. stimulation over the visual cortex (Corthout et al., 2001). M. Sommer et al. / Clinical Neurophysiology 117 (2006) 838–844 843

Maccabee et al. (1998) also reported a biphasic waveform to difference might be due to variations of the pulse be more effective than a monophasic one with regard to configurations obtained with other stimulators (see Fig. 1); threshold of excitation and response amplitude. They offered in addition, in the earlier study, the stimulus intensity steps two possible explanations. One was a role of the initial for SP determination were adjusted to the AMT, which quarter cycle current inducing a hyperpolarization, thereby results in different step width for AP and PA oriented modifying the excitability of voltage-dependent sodium monophasic pulses. channels so as to be more excitable at the subsequent In summary, our data indicate that the stronger asymmetry depolarisation. This is reminiscent of the anode-break of the monophasic pulse results in a more pronounced stimulation explained by Roth (Roth, 1994). Another difference from biphasic pulses than the less asymmetric half explanation was the longer duration of the depolarising sine pulse. In addition, the data suggest a relevance of the 3rd second quarter cycle from the biphasic as compared to the and 4th quarter cycle of the biphasic pulse with regard to the monophasic pulse. Here we show differences between the motor threshold, the MEP latency and the SP duration. The half sine and the biphasic waveform, although the falling difference with regard to the MEP input/output curve further phase in the second quarter cycle of these waveforms is support the idea that mostly monophasic pulses activate a identical. This suggests that the difference between the different subset of cortical interneurons. monophasic and the biphasic waveforms might also involve the 3rd and 4th quarter cycle, and that these quarter cycles lower the motor threshold and reverse the influence of current Acknowledgements direction with regard to motor threshold, MEP latency, and SP duration. A simple mechanism could be the longer M.S. was supported by a DFG grants (So-429/1 and duration of the biphasic as compared to the monophasic or So-429/2), A.A. was supported by a EU Marie Curie the half sine pulse. Hence, our findings make a pivotal role of Training Site Fellowship (HPMT-2001-00413). T.T. was the initial hyperpolarization as suggested by Maccabee et al. supported by the German Ministry for Education and (1998) less likely. Research, Competence Network on Parkinson’s disease In an earlier paper, Brasil-Neto and colleagues studied the (grant 01G10401). We are grateful to an anonymous amplitudes and the latency of MEPs induced in a small hand reviewer who helped to reveal the technical defect that the muscle and systematically varied the orientation offigure-of- MagPro X100 MagOption used in our lab has had. 8 coils over the primary motor cortex (Brasil-Neto et al., 1992). They found an optimal MEP amplitude and the shortest latency with monophasic and biphasic pulses flowing anteriorly in the brain and approximately perpen- References dicular to the presumed location of the central sulcus. Interestingly, for biphasic pulses at higher intensities, the Arai N, Okabe S, Furubayashi T, Terao Y, Yuasa K, Ugawa Y. Comparison between short train, monophasic and biphasic repetitive transcranial orientational specificity was less marked, and another coil magnetic stimulation (rTMS) of the human motor cortex. Clin position yielding high amplitudes appeared about 1808 Neurophysiol 2005;116:605–13 [Epub 2004 Nov 5]. opposite to the first. They concluded that the first and the Bohning DE. Introduction and overview of TMS physics. In: George MS, second phase of the biphasic pulse become effective at higher Belmaker RH, editors. Transcranial magnetic stimulation in neuropsy- intensities. Our data extend these results by showing that the chiatry. Washington, DC: American Psychiatric Press; 2000. p. 13–44. Brasil-Neto JP, Cohen LG, Panizza M, Nilsson J, Roth BJ, Hallett M. current direction is of less pronounced relevance for the half Optimal focal transcranial magnetic activation of the human motor sine and the biphasic pulse than for the monophasic pulse. cortex: effects of coil orientation, shape of the induced current pulse, The results of the SP duration are consistent with a recent and stimulus intensity. J Clin Neurophysiol 1992;9:132–6. study where the authors compared monophasic pulses of Chen R. Interactions between inhibitory and excitatory circuits in the human anterior and posterior current direction with biphasic pulses motor cortex. Exp Brain Res 2004;154:1–10 [Epub 2003 Oct 25]. Corthout E, Barker AT, Cowey A. Transcranial magnetic stimulation. with an initial anterior current direction (Orth and Rothwell, Which part of the current waveform causes the stimulation? Exp Brain 2004). They used however a different set of stimulators, the Res 2001;141:128–32. Magstim 200 for monophasic pulses and the Magstim Super Day BL, Dressler D, Maertens de Noordhout A, Marsden CD, Rapid for biphasic pulses (The Magstim Company, Whit- Nakashima K, Rothwell JC, Thompson PD. Electric and magnetic land, Dyfed, UK). Similar to our data, they found longer SP stimulation of human motor cortex: surface EMG and single motor unit responses [published erratum appears in J Physiol (London) 1990;430: duration with monophasic posteriorly oriented pulses than 617]. 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RESEARCH ARTICLE

Tobias Tings Æ Nicolas Lang Æ Frithjof Tergau Walter Paulus Æ Martin Sommer Orientation-speciﬁc fast rTMS maximizes corticospinal inhibition and facilitation

Received: 11 October 2004 / Accepted: 3 December 2004 / Published online: 3 May 2005 Springer-Verlag 2005

Abstract Specific stimulation of neuronal circuits may Introduction promote selective inhibition or facilitation of corticospinal tract excitability. Monophasic stimulation is more Repetitive transcranial magnetic stimulation (rTMS) likely to achieve direction-specific neuronal excitation. applied over the primary motor cortex is known to In 10 healthy subjects, we compared four types of modulate human corticospinal excitability. While low- repetitive transcranial magnetic stimulation (rTMS), frequency rTMS of 1 Hz can decrease corticospinal monophasic and biphasic stimuli with the initial current excitability (Chen et al. 1997; Muellbacher et al. 2000; in the brain flowing antero-posteriorly (‘‘posteriorly di- Touge et al. 2001; Sommer et al. 2002), high-frequency rected’’) or postero-anteriorly (‘‘anteriorly directed’’). rTMS of 3 Hz or more is generally thought to lead to an We applied rTMS over the primary motor cortex con- increase of corticospinal excitability and thus has a tralateral to the dominant hand, using 80 stimuli at 5 Hz facilitating effect on motor-evoked potentials (MEP) frequency at an intensity yielding baseline motor evoked (Pascual-Leone et al. 1994; Berardelli et al. 1998; Pe- potential (MEP) amplitudes of 1 mV. Monophasic inemann et al. 2000; Romeo et al. 2000; Wu et al. 2000; stimulation was always more efficient than biphasic. Sommer et al. 2001). Different approaches are used to Facilitation was induced by intracerebral anteriorly di- increase or decrease corticospinal excitability selectively. rected current flow and inhibition by posteriorly ori- We here investigate specific stimulus configurations, ented current flow, although only initially for because we found earlier that the inhibitory effect of approximately 30 pulses. The early inhibition was absent low-frequency rTMS depends on the configuration of when studied during a tonic muscle contraction. Several the TMS stimulus. In a paradigm of 15 min sub- mechanisms could account for these findings. They in- threshold 1 Hz rTMS inducing a posteriorly directed clude a more efficient excitation of inhibiting circuits by initial current flow in the brain, monophasic rTMS led posteriorly oriented pulses, and a back-propagating D- to significant inhibition of MEP amplitudes during and wave inhibiting early I-waves and thus inducing early up to 10 min after stimulation, whereas biphasic rTMS inhibition of MEP amplitude. In any case biphasic led to a similar inhibition during stimulation but failed rTMS results can be explained by a mixture of mono- to evoke significant after-effects (Sommer et al. 2002). phasic opposite stimulations. We propose the use of These different effects of monophasic and biphasic monophasic pulses for maximizing effects during rTMS. rTMS on the modulation of corticospinal excitability may account for some of the conflicting results from Keywords Transcranial magnetic stimulation Æ Motor previous studies on the effects of rTMS on measures of cortex Æ Inhibition Æ Facilitation Æ D-waves and I-waves corticospinal excitability and in therapeutic trials utiliz- ing rTMS in the treatment of neuropsychiatric disorders such as major depression, Parkinson’s disease, and epilepsy (Wassermann and Lisanby 2001; Sommer and Paulus 2003). T. Tings Æ N. Lang Æ F. Tergau Æ W. Paulus Æ M. Sommer (&) Based upon this finding we analyzed the effect of Department of Clinical Neurophysiology, high-frequency rTMS on corticospinal excitability to University of Go¨ ttingen, Robert-Koch-Str. 40, obtain further hints about the physiological mechanisms 37075 Go¨ ttingen, Germany underlying the different effects of different stimulating E-mail: [email protected] Tel.: +49-551-396650 conditions. We expected that the pulse configuration Fax: +49-551-398126 may also be important in the modulation of corticospi- 324 nal excitability by high-frequency rTMS and further- muscle between ADM and APB, i.e. the muscle yielding more investigated whether or not the effect of rTMS on the highest amplitudes when stimulated with a chosen corticospinal excitability also depends on the direction intensity, on an individual basis and will refer to it as the of the current flow and on the amount of pre-excitation ‘‘target muscle’’. The remaining hand muscle will be of the motor-system induced by tonic contraction of the referred to as the ‘‘less represented hand muscle’’. The target muscle. position yielding the highest MEP amplitudes for the ‘‘target muscle’’ was determined separately for each of the four types of stimulation, marked with a pen, and Materials and methods the resting motor threshold (RMT) of this muscle was detected by delivering single TMS pulses over the Subjects marked position and reducing the stimulus intensity in steps of 1% stimulator output. The RMT was defined We investigated 18 healthy subjects, 7 women and 11 as the lowest intensity at which at least five out of 10 men (mean age and range 28.8 (20–45) years). None had consecutive MEP induced by test coil stimuli were any neurological or medical disease. The University of ‡50 lV in amplitude with the subject at rest (Rothwell Goettingen ethics committee approved the study, and et al. 1999). The coil was hand held by the investigators written informed consent was obtained from all partic- (TT or MS) throughout the experiment, and the coil ipants. As detailed in the results, eight subjects were position was frequently controlled and corrected, if excluded, resulting in 10 subjects (four women; mean age necessary. and range 29.8 (20–45) years) who completed the principal experiment. Of these, five subjects (two women; mean age 27.6 (22–32) years), participated in control Study design experiment 1, and four subjects (two women; mean age and range 28.8 (22–32) years) also participated in con- Principal experiment trol experiments 2 and 3. In the principal experiment we studied four types of 5 Hz rTMS in four separate sessions in each participant: Experimental set-up monophasic pulses inducing a posteriorly directed initial current flow in the brain, monophasic pulses inducing an During the experiment participants were sitting com- anteriorly directed initial current flow in the brain, bi- fortably in a reclining chair with the arms and neck phasic pulses with posteriorly directed initial current supported. Simultaneous EMG recordings of the flow in the brain, and biphasic pulses with anteriorly abductor digiti minimi (ADM), the abductor pollicis directed initial current flow in the brain. The order was brevis (ABP), the extensor carpi radialis (ECR), and the randomized across subjects and the subjects were blind biceps brachii (BB) muscle on the side of the dominant to the type of stimulation. For the sake of clarity, the hand were sampled at 5 kHz by four pairs of silver– current directions indicated in this manuscript always silver chloride electrodes in a belly-tendon montage, refer to the induced current flow in the brain. filtered at 10 Hz and 2.5 kHz and stored for off-line To determine a baseline of corticospinal excitability, analysis (Synamps, Neuroscan, Herndon, VA, USA). rTMS was preceded by 5 min of single-pulse TMS at a Audiovisual feedback was provided and 50 ms of pre- frequency of 0.1 Hz and at an intensity adjusted to yield TMS EMG was recorded during each trial to control for baseline MEP amplitudes of about 1 mV. Immediately muscle relaxation. Motor threshold and MEP amplitude afterwards, 80 pulses of 5 Hz rTMS were delivered at were assessed before rTMS, and MEP amplitude during the same intensity. This high-frequency rTMS was fol- and after rTMS. lowed by another 10 min of single-pulse TMS at a frequency of 0.1 Hz with unchanged stimulus intensity to detect after-effects (Fig. 1). Transcranial magnetic stimulation Spreading of the facilitatory effects of rTMS applied over the optimum representation of the ‘‘target muscle’’ Monophasic and biphasic TMS pulses were generated to other parts of the motor cortex was monitored by by a MagPro stimulator (initial production series) con- simultaneous recordings from the less represented hand nected to a slightly bent figure-of-eight coil (MC B70, muscle, the ECR, and the BB muscle. Stimulation was Dantec SA, Skovlunde, Denmark). The optimum dom- discontinued if oscillating MEP amplitude patterns oc- inant hand motor representation was determined by curred, or if a lack of relaxation in between pulses was applying strong single TMS pulses with the coil held observed. perpendicular to the presumed localization of the central For analysis, RMT was compared using a repeated- sulcus, with the handle pointing backwards at about 45 measures ANOVA with type of rTMS as within-subjects laterally. To account for possible differences in hand factor. We normalized peak-to peak MEP amplitudes to muscle representation, which might influence the spread the mean corresponding baseline, and we averaged every of excitation, we selected the better-represented hand consecutive six MEP amplitudes recorded before and 325

Fig. 1 Flow chart of experimental procedures

after rTMS, and every consecutive 10 MEP obtained amplitudes during 5 Hz rTMS recorded from the target during 5 Hz stimulation. All mean MEP amplitudes muscle with or without tonic contraction using a paired, were entered in a repeated-measures ANOVA with two-tailed t-test. This analysis was carried out separately muscle and time as within-subjects factors and type of for posteriorly and anteriorly directed rTMS, because rTMS and target muscle (ADM or APB) as between- their difference had been established in the principal subjects factors. Based on significant main effects, we experiment. In addition, we analyzed the MEP latencies compared each bin of MEP during rTMS to baseline of the last 10 baseline MEP and the third bin (21st to the with two-tailed, paired t-tests. 30th MEP) during rTMS. We compared the individual Furthermore, we measured and averaged the MEP mean latencies with those of the principal experiment onset latencies of the third bin during 5 Hz rTMS and using a repeated-measures ANOVA with innervation and compared them with MEP latencies of the last 10 time as within-subjects factors and type of rTMS as baseline MEP. The third bin during rTMS was chosen between-subjects factor. because MEP amplitudes were saturating at that bin, and because it was also available for the control experiment using lateromedial stimulation. We entered Control experiment 2: latero-medial stimulation latencies in a repeated-measures ANOVA with muscle To test whether the strong MEP facilitation observed and time as within-subjects factors and type of rTMS as during monophasic anteriorly directed rTMS is related between-subjects factor. Statview 5.0 (SAS, Cary, NC, to the pattern of D-waves and I-wave recruitment USA) and a significance level of P<0.05 was used for all known to vary according to the coil orientation statistics. (Amassian et al. 1989; Di Lazzaro et al. 2001), we studied rTMS inducing a latero-medial initial current Control experiment 1: rTMS during tonic contraction flow in the brain in four subjects. The set-up was iden- of the target muscle tical with the principal experiment, except that we only tested monophasic pulses in the ‘‘reversed’’ MagPro With five of the participants from the principal experi- current direction mode, and that we rotated the handle ment, we performed two additional sessions that were of the coil 90 laterally (i.e. outwards) of the regular identical with the principal experiment except that par- position. This results in a coil position approximately in ticipants performed a tonic contraction of the target line with the presumed location of the central sulcus, muscle throughout the experiment. Monophasic anteri- thereby inducing medially directed current flow in the orly directed and monophasic posteriorly directed rTMS brain. The optimum ‘‘target muscle’’ representation and were assessed in two separate sessions and data com- the RMT were assessed anew with this coil orientation, pared with the results obtained from the principal because it cannot be inferred from the other orienta- experiment performed with the target muscle at rest. For tions. As corticospinal facilitation had saturated within statistical analysis we compared the normalized MEP 30 pulses in the principal experiment and as we took into 326 account the possibility of a very pronounced rise of (Pascual-Leone et al. 1994) occurred in 8 of the 18 MEP amplitudes that may constitute a safety problem, participants. Marked spreading of excitation to proxi- we reduced the number of 5 Hz rTMS pulses in this mal muscles was also observed for these participants. control experiment to 30. For analysis, we normalized One of these participants reported unpleasant dysaes- MEP amplitudes during rTMS to those before rTMS thesias in the arm contralateral to the stimulation site and compared them with the initial 30 rTMS pulses during monophasic anteriorly directed rTMS and also a from all other types of rTMS recorded in these four sensation of general malaise, which only resolved some subjects during the principal experiment. Furthermore, minutes after premature termination of rTMS. We we measured and averaged the MEP onset latencies of therefore considered 80 pulses of 5 Hz suprathreshold the third bin during 5 Hz rTMS and compared them rTMS to be unsafe for this subgroup of participants and with MEP latencies of the last ten baseline MEP. We excluded them from further participation. entered these subjects’ data from this control experiment In the remaining 10 subjects, no oscillating pattern and from the principal experiment in two separate re- occurred, and complete data with all four rTMS con- peated-measures ANOVA for MEP amplitudes and ditions were obtained without subjective or objective MEP latencies with muscle and time as within-subjects side effects. factors and type of rTMS as between-subjects factor. After having obtained these data, we returned to the subgroup of participants showing oscillating MEP patterns for control experiment 4. Control experiment 3: maximum M-waves amplitudes

To compare the maximum M-wave amplitudes which Resting motor threshold and stimulus intensity can be elicited from the target muscle (APB or ADM) and compare it with the maximum MEP response after The optimum target muscle representation was not rTMS, we elicited M-waves by supramaximum electric identical for all four types of stimuli and differed in the median or ulnar nerve stimulation at the wrist, using a range 0.5–1 cm in each subject. We did not discern a conventional stimulation block as used in nerve con- systematic pattern of ‘‘hot spot’’ variation or an obvious duction studies. Compound muscle action potentials preference for the anterior–posterior or medio-lateral were recorded from the target muscle with a pair of axis. However, we cannot rule out that detailed map- silver–silver chloride electrodes in a belly-tendon mon- ping, which was not the aim of this study, might yield tage. For analysis, we expressed the maximum MEP different centers of gravity (Wassermann et al. 1996) for amplitude during 5 Hz monophasic anteriorly directed the different types of TMS. stimulation as percent of the maximum M-wave elicited The RMT was higher for monophasic than for bi- by peripheral stimulation in each participant. phasic stimuli. The current direction had a marked effect on RMT for monophasic stimuli, where anteriorly directed stimulation resulted in a lower RMT than pos- Control experiment 4: ‘‘inhibitory’’ rTMS in subjects teriorly directed stimulation, and a less pronounced showing oscillating MEP patterns opposite effect for biphasic stimuli, where posteriorly directed stimulation was slightly more effective than Having completed the experiments described above, we anteriorly directed stimulation (effect of type of rTMS, returned to the eight subjects initially excluded for rea- F(3,24)=53.2, P<0.0001; Fig. 2). Post-hoc t-tests reson of oscillating MEP patterns. We sought to detect if vealed a significant difference between all types of the early inhibition of MEP amplitudes during 5 Hz stimulus except for the two biphasic types. rTMS with monophasic posteriorly directed pulses is The stimulus intensity necessary to yield MEP present in this subgroup also. We assumed safety con- amplitudes of 1 mV was significantly different among cerns to be minimal with that particular type of rTMS. rTMS types (effect of type of rTMS, F(3,24)=38.6, Six out of eight subjects could be rescheduled (one lost P<0.0001; Fig. 2). The relative order of stimulus types contact; one refused retesting), and we conducted this was identical with that of RMT, as can be seen in the rTMS condition as in the principal experiment and identical pattern in Fig. 2. Post-hoc t-tests yielded a stopped the intervention at the occurrence of oscillating significant difference between all types of stimulus except patterns. for the two biphasic types. Accordingly, the stimulus intensity relative to the RMT was not different among rTMS conditions (range, Results 119.5–122.6 % of RMT, ANOVA, no effect of type of rTMS, P=0.9). Principal experiment The raw baseline MEP amplitudes of the four muscles studied were similar at each of the four experiments Adverse events done per subject (ANOVA, no effect of session, i.e. of type of rTMS, P=0.28, effect of muscle, F(3,9)=12.6, In the principal experiment, an alternating pattern of P<0.0001). Across all four experiments, MEP baseline low and very high MEP amplitudes as described earlier amplitudes (mean±SD) were 937.5±623.2 lV for the 327

Fig. 2 Resting motor threshold and stimulation intensity. Resting motor threshold (open boxes) and the stimulation intensity (ﬁlled boxes) used before, during and after rTMS are expressed in A lsÀ1, mean±SD (n=10). Note that the pattern is identical for both measures target muscle, 683.3±828.6 lV for the less represented hand muscle, 743.7±735.1 lV for the ECR, and 240.0±311.3 lV for the BB.

MEP amplitudes during rTMS—target muscle

The quantity and direction of change of MEP amplitudes depended on stimulus configuration and on the direction of the induced current in the brain. Mono- phasic stimulation with anteriorly directed initial current flow on average led to a fivefold increase of MEP size with a plateau reached within 4–6 s (i.e. 20–30 stimuli) after the initiation of high-frequency rTMS. In contrast, monophasic stimulation with a posteriorly directed initial current flow did not result in corticospinal facilita- Fig. 3 Typical example of motor evoked potentials recorded in the tion but in fact led to significant inhibition of MEP sizes. target muscle (ADM) of one subject before (B1–B3), during (D1– A typical example is shown in Fig. 3, mean data of all D8), and after (P1–P6) 5 Hz rTMS with monophasic posteriorly directed (left column, A-P) and monophasic anteriorly directed subjects in Fig. 4. (right column, P-A) stimuli. Note that the ordinate scale of the left Biphasic stimulation led to moderate facilitation with a column is twice that of the right column. Fifty milliseconds of twofold to threefold average increase of MEP amplitudes. prestimulus EMG is recorded for confirmation of muscle relaxa- In biphasic stimulation, the amount of facilitation did not tion. Stimulus artifacts are irregularly recorded because of their significantly depend on current direction. The repeated- extremely short duration. Each trace is the average of 10 consecutive recordings (Neuroscan 3.1 averaging software) measures ANOVA on MEP amplitudes yielded an effect of time (F(22,66)=15.0, P<0.001), an effect of type of peculiarities. First, the facilitation extended to the rTMS (F(3,9)=9.4, P=0.001), an interaction of time by proximal muscles, whereas the initial phase of inhibition type of rTMS (F(66,770)=6.0, P<0.001), no main effect of within the first bins of 5 Hz monophasic posteriorly muscle, no main effect of target muscle, and no interaction directed rTMS was present in the distal muscles only of target muscle with time, and no other significant inter- (Fig. 4). The absence of inhibition in the proximal actions. Planned post hoc t-tests of MEP during rTMS muscles is unlikely to be a ‘‘floor’’ effect because of the revealed significant differences between monophasic large baseline amplitudes at least for the ECR. Second, anteriorly directed rTMS and all other types of rTMS. For post-hoc paired t-tests across all normalized bins during the target muscle, post hoc paired t-tests across all bins rTMS indicated that the MEP facilitation for mono- during rTMS revealed a significant difference between all phasic anteriorly oriented rTMS was significantly types of rTMS except between the two biphasic types. stronger in the target muscle than in all other muscles, whereas the facilitation with biphasic posteriorly direc- MEP amplitudes during rTMS—other muscles ted rTMS the facilitation was significantly stronger in the less represented hand muscle than in the target The overall pattern described for the target muscle was muscle and the BB. For the biphasic anteriorly directed present in the other muscles also, but there were three rTMS, also, MEP facilitation in the less represented 328

Fig. 4 Principal experiment—MEP amplitudes. MEP amplitudes before, during, and after 5 Hz rTMS of four different stimulus types indicated by different symbols, simultaneously recorded in four different arm muscles (A the better represented muscle among abductor pollicis brevis and abductor digiti minimi, B the less represented hand muscle, C extensor carpi radialis, and D biceps brachii). Mean±SE. At baseline, 30 pulses were given at 0.1 Hz frequency. During rTMS, 80 pulses at 5 Hz were applied, and the resulting MEP grouped in bins of 10 each. After rTMS, the baseline was repeated twice. Asterisk, significant difference from baseline in post-hoc t-test (P<0.05)

hand muscle was significantly stronger than in any other 24.3±1.6 ms for biphasic anteriorly directed stimula- muscle. Third, in the more proximal muscles, the effects tion, and 22.7±1.5 ms for biphasic posteriorly directed of the biphasic types of rTMS tended to be dissociated. stimulation (effect of type of rTMS, F(3,26)=4.7, Biphasic posteriorly directed stimuli yielded a facilita- P=0.008). There were no significant interactions. tion in ECR and BB, biphasic anteriorly directed stimuli induced much less facilitation in the ECR and no significant change of MEP amplitude in the BB (Fig. 4). Control experiment 1: rTMS during tonic contraction of the target muscle

Effects outlasting rTMS When the principal experiment was repeated in a subgroup of patients with MEP recorded during tonic With all four stimulation procedures, MEP amplitudes contraction of the target muscle (stimulus intensity again returned to baseline level within 1 min after the end of adjusted to yield baseline potentials of 1 mV from the high-frequency rTMS, and there were no significant target muscle), posteriorly directed monophasic 5 Hz after effects (no effect of time or type of rTMS after rTMS in contrast to the principal experiment did not rTMS). lead to inhibition of MEP size but to a moderate, about twofold facilitation (t-test, P<0.001, Fig. 5). The facil- MEP latencies itatory effect of anteriorly directed monophasic rTMS was essentially unaffected by the tonic contraction of the The MEP latencies were longer with distal than with target muscle (t-test, P=0.52). proximal muscles and shortened during rTMS (re- Voluntary muscle contraction had an effect on the peated-measures ANOVA, effect of muscle, MEP latency (effect of innervation, F(1,1)=7.3, F(3,9)=124.0, P<0.0001, effect of time, F(1,3)=5.6, P=0.027). For example, in the target muscle the base- P=0.025; target muscle MEP latency shortening of less line MEP onset latency (mean±SD) was 21.4±1.2 ms than 0.3 ms for each type of rTMS). MEP latencies were (at rest, 22.9±1.4 ms) for monophasic anteriorly direc- differentiated according to the stimulus type. For ted stimulation during voluntary contraction. For example, in the target muscle the baseline MEP onset monophasic posteriorly directed stimulation during latency (mean±SD) was 23.7±1.9 ms for monophasic voluntary contraction it was 23.0±0.9 (at rest, anteriorly directed stimulation, 24.6±1.7 ms for 24.1±1.4 ms). There was a trend for an effect of type of monophasic posteriorly directed stimulation, rTMS (F(1,8)=4.3, P=0.071), with anteriorly oriented 329

during 5 Hz monophasic anteriorly directed rTMS, i.e. the stimulus condition leading to the strongest MEP facilitation. The maximum MEP amplitude obtained during 5 Hz monophasic anteriorly directed rTMS did not exceed one third of the maximum M-wave amplitude in any subject (mean±SD 16.2±11.2%).

Control experiment 4: ‘‘inhibitory’’ rTMS in subjects showing oscillating MEP patterns

In all six of the eight subjects initially excluded for reason of oscillating MEP patterns the two initial bins of MEP amplitudes during rTMS could be completed. They yielded an MEP amplitude normalized to baseline of 1.22±0.90 in bin 1 and 2.09±1.67 in bin 2 (mean±SD).

Discussion

Fig. 5 Control experiment 1. rTMS during tonic contraction of the These results dissociate inhibitory and facilitatory target muscle. MEP amplitudes during and after monophasic 5 Hz mechanisms behind rTMS of a given stimulation fre- rTMS with anteriorly and posteriorly directed current flow in the quency by showing that both inhibition and facilitation brain and recorded from the target muscle at rest (filled symbols) can be achieved by 5 Hz rTMS, and that the net effect of and from the target muscle under tonic contraction (open symbols). n=5, mean±SE 5 Hz rTMS on corticospinal excitability depends on the configuration of the TMS-pulse, on the direction of the pulses yielding shorter latencies. There were no signifi- induced current, and on the state of excitation of the cant interactions. sensorimotor system at the time of application of rTMS. In the past, the stimulation frequency has been regarded as the single most important factor determining Control experiment 2: latero-medial stimulation whether a train of rTMS leads to an increase or a de- In the four subjects studied in this control experiment, crease of corticospinal excitability. One Hertz has been the mean RMT for monophasic medially directed pulses regarded a the gold standard of a watershed for differ- was 97.0±3.6 A lsÀ1 and thus higher than for stimu- entiating between inhibition and facilitation by many lation with any other condition. The mean target muscle researchers with rTMS-frequencies at or below 1 Hz MEP latency at baseline (23.0±1.94 ms) was at the leading to decreased corticospinal excitability and higher lower end of the range of the latencies obtained with the other stimulation conditions, which were not substantially different from the principal experiment. Monophasic pulses with medially directed current direction induced an MEP amplitude facilitation during rTMS similar to that observed during biphasic stimulation of both directions. In all four subjects studied, it was less pronounced than the facilitation observed with anteriorly directed monophasic stimulation (no main effect of type of rTMS, main effect of time, F(7,28)=9.2, P<0.001; interaction of time by type of rTMS, F(28,105)=1.7, P=0.034; no effect of muscle, no interaction of muscle by time or muscle by type of rTMS; Fig. 6). Post hoc tests yielded a significant difference only between monophasic anteriorly directed and monophasic posteriorly directed rTMS.

Control experiment 3: maximum M-waves Fig. 6 Latero-medial rTMS. MEP amplitudes before and during rTMS in four subjects, mean±SE. At baseline, 30 pulses were given at 0.1 Hz frequency and grouped in bins of six pulses each. In four subjects, M-wave amplitudes after supramaximal During rTMS, 30 pulses at 5 Hz were applied, and the resulting peripheral electric stimulation were measured and com- MEP grouped in bins of 10 each. Symbols reflect different TMS pared with the amplitude of the largest bin of MEP pulses as indicated in the legend 330 rTMS frequencies generally leading to an increase of subthreshold–suprathreshold pairs of TMS (Sommer corticospinal excitability and thus facilitation of MEP. et al. 2001). This is similar to this study in which inhi- However, recently it was shown that 1 Hz rTMS can bition with monophasic posteriorly directed pulses was also be facilitatory and 5 Hz rTMS can be inhibitory not sustained throughout the duration of rTMS. Two depending on the state of cortical excitability at the time other points however dissociate intracortical condition- (Siebner et al. 2004; Lang et al. 2004). This indicated ing-test paired-pulse inhibition from what we observed that the ‘‘classical’’ view of pure frequency-dependence here. First, circuits responsible for intracortical inhibi- of rTMS effects may be too simplistic. tion are not sensitive to the direction of the conditioning The fact that in our study anteriorly directed mono- TMS pulse (Ziemann et al. 1996), but our data suggest a phasic currents induce corticospinal facilitation whereas high orientational specificity of the inhibitory circuits posteriorly directed monophasic currents lead to corti- involved. Second, the topography of facilitation and cospinal inhibition suggests that there is differential inhibition differs. In our study facilitation was observed activation of intracortical circuits depending on the in all muscles studied, whereas inhibition was limited to direction of the current flow in the brain, which then the hand muscles. This pattern is in contrast with the sum up to an inhibitory or facilitatory net effect of topography of intracortical inhibition and intracortical rTMS. This interpretation is in line with previous facilitation as tested with a conditioning-test paired- experiments with a similar setup using 1 Hz subthresh- pulse TMS procedure (Kujirai et al. 1993; Ziemann et al. old stimulation. In these experiments, all four stimula- 1996), because with that paradigm the area for inhibition types (i.e. biphasic and monophasic rTMS with tion is larger than that for facilitation (Ashby et al. anteriorly or posteriorly directed current flow) led to 1999). It is therefore unlikely that the circuits responsible acute inhibition of MEP size as expected but only the for rTMS-induced MEP modulation are identical with monophasic posteriorly directed currents yielded a sig- those responsible for intracortical excitability. nificant MEP inhibition outlasting the rTMS over the An alternative explanation is motivated by corti- primary motor cortex (Sommer et al. 2002, unpublished cospinal tract recordings in patients with spinal epidural observations). Therefore it seems that monophasic pos- electrodes. These studies showed that a corticospinal teriorly directed rTMS leads to a preferential activation volley consists of an early D-wave, thought to result of inhibitory circuits and therefore induces corticospinal from direct activation of corticospinal axons, and later I- inhibition even when used within an otherwise facili- waves, numbered consecutively and thought to be gen- tating 5 Hz paradigm and induces prolonged corti- erated transsynaptically (Patten and Amassian 1954; cospinal inhibition in an inhibitory 1 Hz paradigm. Ziemann and Rothwell 2000). Anodal transcranial electrical stimulation (TES), and in some subjects also latero-medially oriented monophasic TMS (Di Lazzaro Possible mechanisms et al. 2003), evoke an early D-wave (Rothwell et al. 1991). With a circular coil centered over the vertex (Di There are several possible explanations of our findings. Lazzaro et al. 2002b), or monophasic posteriorly ori- The preferential activation of inhibitory circuits with ented pulses from a figure-of-eight coil (Di Lazzaro et al. posteriorly oriented currents is consistent with a recent 2001), a D-wave with a slightly (0.2 ms) longer latency study on the duration of the cortical silent period (cSP) is observed. This delay suggests a more proximal site of induced by monophasic anteriorly directed pulses, corticospinal tract stimulation, possibly at the first in- monophasic posteriorly directed pulses and biphasic ternodes or close to the axon hillock (Di Lazzaro et al. pulses with an initial anteriorly directed current flow 2001). When such a delayed D-wave is observed, the (Orth and Rothwell 2004). They found that the duration early I-waves may be suppressed, probably because of of the cSP was shorter with monophasic anteriorly di- back-propagating excitation of the pyramidal tract ax- rected stimulation than with monophasic posteriorly ons stimulated close to the axon hillock (Di Lazzaro directed pulses. The authors suggested this may reflect a et al. 2002b, 2004), resulting in a refractoriness of the less efficient excitation of inhibitory intracortical circuits pyramidal tract neuron. The I-1 waves are preferentially by monophasic anteriorly directed TMS. If the same was observed with monophasic anteriorly directed stimula- true for other, longer lasting intracortical inhibitory tion, later I-waves are preferentially observed with circuits, such as for the long-interval intracortical inhi- monophasic posteriorly directed stimulation (Di Lazz- bition (LICI, Chen 2004), i.e. if the LICI lasted longer aro et al. 2001). To our knowledge no epidural record- with monophasic posteriorly oriented pulses than with ings during monophasic rTMS have been studied (Di anteriorly oriented pulses, then the corticospinal motor Lazzaro et al. 2002a). system would be in part refractory when a second pulse Even though the selectivity of D-wave and I-wave is delivered 200 ms after a first one. We are however not elicitation might be less pronounced with relatively aware of an LICI study comparing different current strong stimuli as used here, we hypothesize that rTMS directions. with a technique evoking a ‘‘delayed’’ D-wave will With regard to circuits mediating short-interval in- determine an increase in this wave but this will be par- tracortical inhibition, we have reported a progressive alleled by an inhibition of the early I-waves due to the loss of inhibition during 5 Hz rTMS with inhibitory antidromic activation of the corticospinal axons. As 331 more than one descending volley is required to evoke an known, it could be related to the stimulation of partially MEP at rest (Di Lazzaro et al. 1998), the net effect could different neuronal subpopulations (Di Lazzaro et al. be inhibition, at least early during a train of repetitive 2001). For biphasic pulses, this order was seemingly stimuli. On the other hand, repetitive stimulation with a reversed. This results support the notion that the second technique that evokes preferentially early I-waves will and third quarter cycle of a biphasic pulse, inducing achieve maximum facilitation, because rTMS with this currents oppositely directed to those induced by the technique will preferentially increase the I-wave ampli- initial quarter cycle, are the decisive components in tudes, which explains why anteriorly directed rTMS terms of MEP elicitation (Maccabee et al. 1998). leads to marked facilitation in our study. During vol- The effect of voluntary contraction was not sub- untary contraction one single descending wave is capa- stantially different for monophasic anteriorly than for ble of evoking an MEP, and I-waves are facilitated (Di monophasic posteriorly directed currents in the subjects Lazzaro et al. 1998). Therefore, posteriorly directed of control experiment 1. This does not enable us to draw rTMS results in facilitation of MEP evoked during clear conclusions as to the sites stimulated by either voluntary contraction. The latero-medial rTMS in four pulse (Di Lazzaro et al. 2001). subjects yielded MEP latencies not substantially shorter than other types of TMS, suggesting that D-wave elicitation might not have been successful in all subjects. Cortical or subcortical effects? This is consistent with earlier findings (Sakai et al. 1997). We cannot rule out yet another alternative explana- It has been shown by others that rTMS at 5 Hz can tion, which is that the mechanism underlying the facili- clearly modify cortical excitability (Di Lazzaro et al. tatory effect of anteriorly directed rTMS is based upon 2002a). Because we had to use suprathreshold pulses to the gradual pulse-to-pulse depolarization of a trans- be able to measure the effects during rTMS, we have to membrane potential which would be uniformly achieved consider spinal effects also. In our set of data there is at by selective and asymmetric monophasic pulses. The least indirect evidence that makes a predominantly inhibition observed with monophasic posteriorly direc- subcortical origin rather unlikely. First, subjects with an ted stimulation might be interpreted as a hyperpolar- oscillating MEP amplitude pattern, which indicates a ization achieved with oppositely directed, asymmetric synchronization of spinal motor neurons (Pascual- currents. Following this train of thought, the different Leone et al. 1994), had been excluded from the primary active components of biphasic pulses would induce a data set and, in fact, these subjects failed to show an mixed effect of depolarization and hyperpolarization early MEP inhibition with monophasic posteriorly ori- counteracting each other and thus limit the net effect of ented rTMS (control experiment 4). Second, in the 10 biphasic rTMS to the difference between its facilitatory subjects without oscillating MEP amplitudes the facili- and inhibitory compounds. The absence of lasting effects tation observed with monophasic anteriorly directed does not necessarily rule out this hypothesis, because the stimulation saturated well before the end of rTMS at a duration of stimulation may have been too short. It level considerably below the maximum M-wave, with no should be noted, however, that much of the animal lit- further increase during the second half of rTMS (control erature in this regard (Bartlett et al. 1977; Girvin 1978) experiment 3). This again is in contrast with the MEP deals with stronger and longer lasting currents and is steadily increasing during stronger and faster rTMS and therefore not directly applicable here. approaching the amplitude of the peripheral M-wave Taken together, the mechanism behind our results observed with spinal motor neuron synchronization cannot be elucidated with the methods used here. A (Pascual-Leone et al. 1994). Third, the shortening of study on LICI should yield insight with regard to the MEP latencies during rTMS compared with baseline first hypothesis, epidural recordings with regard to the was less than 0.3 ms for all types of stimulation. This is second. Whatever explanation holds true, the results of less than described for the latency shift of 1–2 ms with biphasic stimuli can be readily explained by a mixture of subcortical stimuli induced by TES or transcranial monophasic opposite stimulations canceling each others’ magnetic medially directed stimulation (Rothwell 1991). specific effects. We conclude that the different compo- It would have been desirable to use F-wave or H- nents of biphasic pulses were not at all synergistic with reflex measurements or TES to further explore the regard to MEP facilitation, and that the more orienta- contribution of subcortical circuits on the observed re- tion specific monophasic pulses exploit a wider range of sults. However, as TES, F-wave and H-reflex measure- corticospinal excitability changes. ments depend, in fact, on somewhat painful electrical stimuli, it is not possible to intermingle them with TMS pulses at 5 Hz without significantly influencing the level Latency of relaxation of the participant and thus disturbing the recording. As there are no significant after-effects in ei- In our study, anteriorly directed monophasic pulses ther the main experiment or any of the control experi- yielded shorter latencies than posteriorly directed pulses. ments, assessing excitability changes on a subcortical Our findings are consistent with results from Di Lazzaro level by means of TES, F-waves or H-reflexes carried out et al. (2001). The origin of this latency difference is not immediately before and after 5 Hz rTMS would not be a 332 meaningful control experiment either. Therefore, while the above considerations make a substantial subcortical Conclusion contribution unlikely, definite proof would require invasive recordings from epidural cervical space. In conclusion, the marked difference between both types of monophasic rTMS indicates that one may selectively induce corticospinal inhibition or facilitation with 5 Hz Problem of stimulus intensity rTMS. If the current direction is appropriately chosen, monophasic rTMS has the potential to yield more Monophasic posteriorly directed TMS has the highest selective and stronger effects than seen with biphasic RMT of the four conditions studied and requires the rTMS of either current direction. On the way to opti- highest stimulation intensity, as reported earlier (Nie- mizing effects in terms of specificity and duration apart haus et al. 2000; Kammer et al. 2001). We cannot rule from other techniques (tDCS (Nitsche and Paulus 2000), out that the inhibition obtained during rTMS is an theta burst (Huang et al. 2004)) this finding may be epiphenomenon of different stimulus intensities, with relevant for future studies, at least for those inducing higher absolute stimulus intensities leading to inhibition immediate effects on corticospinal excitability. First, rather than facilitation. However, ordering the rTMS effects during rTMS, such as the induction of functional conditions according to the inhibition or facilitation lesions, might profit from an appropriate choice of the induced during rTMS results in a different pattern from rTMS type. Second, one may need to redefine the that of the stimulus intensities (Fig. 2). This pattern of established safety criteria (Wassermann 1998) to account stimulus intensities is clouded by the technical limitation for the influence of stimulus configuration and current that the current indicator of the Dantec MagPro stim- direction on the immediate effects of rTMS. ulator only measures the first 50 ls of a magnetic pulse (Dr Kienle, Medtronic, personal communication). Acknowledgements We are grateful to an anonymous reviewer for Therefore, the biphasic threshold values indicated by the contributing one of the hypotheses to explain these data, and to M.A. Nitsche and A. Antal for fruitful discussion. Part of this work stimulator are probably artificially low, because the has previously been presented in abstract form (Sommer et al. 2003; falling phase of a biphasic pulse (second and third Tings et al. 2004). M.S. was supported by the DFG (Deutsche quarter cycle) is not taken into account. It is, therefore, Forschungsgemeinschaft) Grant SO 429/2-1, W.P. by the DFG not possible to clarify the role of the stimulation inten- Graduiertenkolleg GRK 632. sity with present stimulator systems in the motor cortex. Reducing the intensity of monophasic posteriorly directed stimuli to the intensity used for biphasic pulses to References elicit an MEP of 1 mV results in stimuli not sufficient to evoke a MEP response. Increasing the intensity of Amassian VE, Cracco RQ, Maccabee PJ (1989) Focal stimulation monophasic anteriorly oriented pulses or of biphasic of human cerebral cortex with the magnetic coil: a comparison pulses of either direction to the absolute intensity as used with electrical stimulation. 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J Neu- repetitive transcranial magnetic stimulation on intracortical rophysiol 17:345–363 excitability in human subjects. Neurosci Lett 287:37–40 Peinemann A, Lehner C, Mentschel C, Munchau A, Conrad B, Ziemann U, Rothwell JC (2000) I-waves in motor cortex. J Clin Siebner HR (2000) Subthreshold 5-Hz repetitive transcranial Neurophysiol 17:397–405 magnetic stimulation of the human primary motor cortex re- Ziemann U, Rothwell JC, Ridding MC (1996) Interaction between duces intracortical paired-pulse inhibition. Neurosci Lett 15:21– intracortical inhibition and facilitation in human motor cortex. 24 J Physiol 496:873–881 White Matter Asymmetry in the Human C. Büchel1, T. Raedler2, M. Sommer3, M. Sach1, C. Weiller1 and M.A. Koch1 Brain: A Diffusion Tensor MRI Study 1NeuroImage Nord, Department of Neurology, Hamburg University Medical School, Germany, 2NeuroImage Nord, Department of Psychiatry, Hamburg University Medical School, Germany and 3Department of Clinical Neurophysiology, University of Göttingen, Germany

Language ability and handedness are likely to be associated with sion-tensor MR imaging has also indicated subtle white matter asymmetry of the cerebral cortex (grey matter) and connectivity abnormalities in developmental speech (Sommer et al., 2002) (white matter). Grey matter asymmetry, most likely linked to and language disorders (Klingberg et al., 2000). language has been identified with voxel-based morphometry (VBM) Voxel-based morphometry (VBM) studies investigating brain using T1-weighted images. Differences in white matter obtained with asymmetry have focused on grey matter (Watkins et al., 2001). this technique are less consistent, probably due to the relative insen- One study also investigated differences in white matter using sitivity of the T1 contrast to the ultrastructure of white matter. T1-weighted images (Good et al., 2001). In the right hemi- Furthermore, previous VBM studies failed to find differences related sphere, higher grey matter density was found in the frontal to handedness in either grey or white matter. We revisited these lobe, whereas the opposite was found in the occipital and the issues and investigated two independent groups of subjects with anterior temporal lobe in accord with previously described diffusion-tensor imaging (DTI) for asymmetries in white matter larger indentations of the skull (i.e. petalia). Interestingly, this composition. Using voxel-based statistical analyses an asymmetry of does not seem to reflect the marked difference in lateralization the arcuate fascicle was observed, with higher fractional anisotropy of language to the left hemisphere, according to which one in the left hemisphere. In addition, we show differences related to would expect higher grey matter density in left fronto- handedness in the white matter underneath the precentral gyrus temporal areas. Similarly, based on the profound differences in contralateral to the dominant hand. Remarkably, these findings were motor skills between the dominant and non-dominant hands, very robust, even when investigating small groups of subjects. This one would expect marked differences in the organization of highlights the sensitivity of DTI for white matter tissue differences, the motor system depending on the handedness of the subject. making it an ideal tool to study small patient populations. However, the two studies using VBM found no such difference when investigating either grey matter (Watkins et al., 2001) or Keywords: connectivity, DTI, handedness, language, morphometry, MRI, grey and white matter (Good et al., 2001). So far, only studies white matter investigating the depth of the central sulcus revealed a left–right asymmetry related to handedness in males but not in females (Amunts et al., 2000). The negative result of these VBM Introduction studies is unlikely to be related to insufficient statistical power,

Voxel-based statistical analysis is usually performed on T1- as both studies investigated >100 subjects. This is in marked weighted magnetic resonance (MR) images and has revealed contrast to traditional morphometry studies that have consist- details of anatomical differences in vivo (Ashburner and ently revealed a correlation between the asymmetry in the Friston, 2000; Good et al., 2001). Studies have shown changes planum temporale and planum parietale and hand dominance in grey matter density related to navigational skills (Maguire et (Steinmetz et al., 1991; Jäncke et al., 1994). This leaves the al., 2000), specific forms of headache (May et al., 1999) and possibility that signal intensity in T1-weighted images is an degenerative diseases (Baron et al., 2001). insensitive marker for anatomical differences related to hand- A problem concerning white matter morphometry on the edness. basis of T1-weighted images is that T1 signal intensities are not We revisited this issue and investigated two independent very well correlated to white matter integrity. This can be seen groups of healthy subjects with diffusion-tensor imaging (DTI) in disorders such as multiple sclerosis, in which white matter and voxel-based statistical analysis to further investigate the

T1 signal intensities remain normal even in severely damaged following questions: (i) are there differences between the tissue (Filippi et al., 2001). ultrastructure of white matter in the left versus the right hemi-

In contrast to T1-weighted imaging, diffusion-weighted sphere? and (ii) is handedness reflected by a different white imaging provides more subtle information about white matter matter composition in the hemisphere contralateral to the tissue composition (Basser, 1995). Diffusion tensor (magnetic dominant hand? resonance) imaging (DTI) can be used to measure the diffusion characteristics of water in vivo. White matter fiber orientation Materials and Methods can be determined by using diffusion-tensor MR imaging because water diffuses faster parallel to the longitudinal axis of Subjects axons than perpendicular to it (Basser et al., 1994). The frac- We studied two groups of subjects with DTI. The first group consisted of 15 healthy volunteers (mean 30.0, range 23–43 years, four females) tional anisotropy (FA) of diffusion is sensitive the coherence of and was previously used as a healthy control group for a sample of the orientation of fibers within each voxel. Lower FA values stutterers (Sommer et al., 2002). Most of the volunteers were can indicate decreased fiber coherence or myelination defects employees of the university hospital. None of the participants as observed in multiple sclerosis (Filippi et al., 2001). Diffu- suffered from any neurological or unstable medical disease or took

Cerebral Cortex V 14 N 9 © Oxford University Press 2004; all rights reserved Cerebral Cortex September 2004;14:945–951; doi:10.1093/cercor/bhh055 any CNS-active medication. All volunteers in this group were subjects between hemispheres using SPM2 (paired t-test) with a consistent right handers with a score of at least 15/22 points threshold set to P < 0.05, corrected for multiple comparisons. For the (Oldfield, 1971). handedness analysis, we hypothesized increased FA contralateral to The second group consisted of 28 healthy volunteers (mean 29.9, the dominant hand in motor/premotor areas. In this case the correc- range 21–40 years, 14 females). Nine subjects were left handed, 19 tion of the P-value was based on a volume of interest (sphere with right handed. The mean handedness score on the 10-item version of radius 12 mm, i. e. a volume of 7400 mm3) centered around the hand the Edinburgh Handedness Inventory (Oldfield, 1971), calculated as knob (Yousry et al., 1997). 100*((R – L)/(R + L), was 86.8 ± 15.9 (range 53.8–100) for the right handers and –79.7 ± 17.2 (range –60 to –100) for the left handed Eigenvector Visualization volunteers. Right handers were on average 29.7 ± 5.5 years old and To visualize differences between hemispheres in an anatomical left handers were 30.3 ± 6.5 years old. There was no significant age context of fiber bundles, we used a common scheme to represent the difference between groups [F(1,26) = 0.1, P = 0.8]. Both studies were orientation of the first eigenvector (i.e. the eigenvector corre- approved by the local ethics committee and subjects gave written sponding to the largest eigenvalue) with colors. The magnitude of the informed consent prior to the experiment. Cartesian components (x, y, z) of this vector are color-coded: |x| (left–right) in red, |y| (posterior–anterior) in green and |z| (infe- Image Acquisition rior–superior) in blue. Note that this simple representation is ambig- Diffusion-weighted images were acquired on a Magnetom Vision 1.5 T uous: in the worst case two perpendicular vector orientations are MR system (Siemens Erlangen, Germany) with a circularly polarized displayed in the same color (see, for example, Pajevic and Pierpaoli, head coil and maximum gradient amplitude of 25 mT/m. Cushions 1999). All color plots were derived from a high resolution (2 × 2 × were used to restrict the subject’s head movements. The participants 3 mm voxel-size) template DTI scan of a healthy control subject, as wore earplugs for noise protection. We acquired diffusion-weighted previously reported (Koch et al., 2002). images with a ‘stimulated echo acquisition mode’ (STEAM; Nolte et al. ° T T × , 2000) sequence (flip angle = 15 , R = 8872 ms, E = 65 ms, 56 64 Results matrix, field of view = 168 × 192 mm, voxel size = 3 × 3 × 5 mm3) of 20 slices covering the whole brain and parts of the cerebellum. The Asymmetry full protocol consisted of a T2-weighted image and six diffusion- weighted images sensitized for diffusion along six different directions To study the reliability of our approach, hemispheric asym- (b-value = 750 s/mm2). These measurements were repeated 40 times metry was assessed for both groups of volunteers individually. to improve the signal-to-noise ratio in the tensor maps. A T1-weighted In group 1, testing for greater FA in the left than the right image was acquired using a 3-D ‘fast low angle shot’ sequence (flip hemisphere revealed a C-shaped structure connecting the ° T T × × angle = 30 , R = 15 ms, E = 5 ms, 256 256 196 matrix, voxel size temporal and frontal cortex. This structure comprised the = 1 × 1 × 1 mm3). posterior part of the superior longitudinal fascicule, a part of Image Processing and Statistical Analysis the arcuate fascicle. The observed FA difference extended far The first diffusion-weighted image was discarded to exclude the tran- into the temporal lobe (Fig. 1, top left). Performing the same sition to steady state. Image processing was performed with SPM 99 analysis in group 2 revealed an identical pattern of differences and SPM2 (http://www.fil.ion.ucl.ac.uk/spm/). The diffusion- with significantly greater FA the arcuate fascicle of the left weighted images were realigned to the second image without diffu- hemisphere (Fig. 1, top right). Overlaying this activation onto sion weighting, and co-registered with the high resolution T image, 1 a color-coded direction map revealed that the difference was which we then spatially normalized to a standard template (Paus et al., 1999), reorienting the diffusion gradient directions accordingly. The confined to areas in which fibers are predominantly oriented DTI images were sinc interpolated to 2 × 2 × 2 mm3 resolution. The anterior–posterior (green in Fig. 1, top left). The same was true diffusion-tensor and fractional anisotropy (FA) were determined for for the second group (Fig. 1, top right). every voxel according to standard methods (Basser et al., 1994). Testing for greater FA in the right than in the left hemisphere Corrections for eddy current distortions were not necessary for this revealed a smaller area of increased FA posterior to the arcuate STEAM DTI acquisition. fascicle in the white matter underneath the inferior parietal To account for different geometrical configuration of both hemispheres, we performed two additional normalization steps: In a first cortex, just above the Sylvian fissure (Fig. 1, bottom left). An step, all FA volumes were averaged to create a mean FA image. This identical difference was observed in the second group (Fig. 1, image was then averaged with a mirrored version of itself, generating bottom right). The overlay on color coded direction maps a symmetrical FA template. We then spatially normalized all FA showed this difference to be located in an area in which the volumes to this symmetrical template. predominant direction of diffusion is in the lateral direction It is conceivable that both hemispheres are not perfectly parallel (i.e. right–left). (e.g. smaller interhemispheric gap between the occipital lobes). If the relative position between left and right hemisphere in an individual brain is different from that in the template, spatial normalization can Handedness shift the brain slightly to one side, which renders spatially normalized Differences in fractional anisotropy in relation to handedness FA maps erroneously asymmetric. To avoid these artifacts, in a second were only assessed for the second group of subjects in which 9 step the hemispheres were separated, all right hemispheres flipped to of 28 subjects were left handed (Fig. 2). Here we tested for the left and individual hemispheres spatially normalized to the left greater FA in the right as compared to the left hemisphere for hemisphere of the symmetrical FA template. left handers and greater FA in the left as compared to the right We used a 12 parameter affine registration together with non-linear warps parameterized through a basis set of discrete cosines (7 × 8 × 7 hemisphere for right handers. This was done using a conjunc- cycles in x, y and z directions) in all normalization procedures. The tion analysis, reporting the minimum t-value of both compari- non-linear step was considered important, since we wanted to register sons (Friston et al., 1999). In this analysis only one significant different brains and hemispheres as accurately as possible. This is in difference (Z = 4.0, P < 0.05 corrected for ) was observed in the contrast to standard VBM approaches, in which a residual difference frontal lobe (x = ±32, y = –14, z = 50 mm; Fig. 2c). Closer in structure is desired (Ashburner and Friston, 2000). These rather inspection revealed that this signal difference is located exactly complicated procedures were necessary to avoid possible confounds of differential registration of the two hemispheres. within the white matter of the precentral gyrus. Both pre- and Finally, FA maps were smoothed with a Gaussian kernel of 4 or 12 postcentral gyri are easily identified on FA images by their high mm full width at half maximum. FA values were compared within fractional anisotropy value (arrows in Fig. 2a). In this area FA

946 DTI of White Matter Asymmetry • Büchel et al. Figure 1. Differences in fractional anisotropy between hemispheres thresholded at P < 0.05 (corrected for multiple comparisons). Columns indicate groups, i.e. independent data samples from two groups of volunteers and rows indicate the comparison, either higher FA on the right than left (lower rows) or vice versa (upper rows). The top left quadrant shows significantly higher FA in the arcuate fascicle of the left hemisphere. Overlaying this difference on a color coded orientation scheme revealed that the fibers in this area of the arcuate fascicle have a predominant anterior-posterior orientation. An almost identical picture emerged in group 2 (top right quadrant). See Supplementary Material for a color version of this figure. The bottom row shows higher FA in the right hemisphere in the vicinity of the corpus callosum in the white matter of the parietal lobe. This was replicated in group 2 (top right quadrant). Note that images in the bottom row are mirrored across the y–z plane (i.e. R and L are reversed). was consistently higher in the left compared to the right hemi- ability showing an identical pattern in two independent sphere in right handers and vice versa in left handers (Fig. 2b). samples. Furthermore, a detailed analysis of handedness revealed a significant greater FA in the precentral gyrus contral- Discussion ateral to the dominant hand. Using voxel-based statistics on fractional anisotropy maps Recent voxel-based morphometric studies investigating derived from DTI we were able to show greater FA values in brain asymmetry focused on the grey matter partition. The the arcuate fascicle in the left hemisphere and in the inferior most prominent finding was a higher grey matter density in the parietal white matter in the right hemisphere. The robustness left planum temporale (Good et al., 2001; Watkins et al., 2001). of this result was demonstrated by an excellent test–retest reli- Interestingly, this area of increased grey matter density is close

Cerebral Cortex September 2004, V 14 N 9 947 Figure 2. Differences in fractional anisotropy related to handedness thresholded at P < 0.001. Handedness-related higher FA was found in the white matter of the precentral gyrus (a). The central sulcus can be easily identified on FA images since it is bordered by the precentral and postcentral gyri, which can easily be identified in FA images. FA values in this area are significantly higher in the left as opposed to the right hemisphere for right handers and vice versa for left handers (b). Bars (±90% confidence intervals) indicate average FA difference between hemispheres within group (left- or right handers). This finding was highly selective as is shown on the maximum intensity projection (c). See Supplementary Material for a color version of this figure. to the region of FA difference between right and left hemi- data were normalized to a symmetric template that was derived spheres which was observed in our study. Only one study from the group studied. It is conceivable that although both directly investigated asymmetry of ‘white matter density’ as hemispheres are congruent, they may not be perfectly parallel, estimated from T1-weighted images (Good et al., 2001). The e.g. due to a slightly smaller interhemispheric gap between the extensive asymmetries reported in this study partially included occipital lobes than between the frontal lobes. If the relative the arcuate fascicle. position between left and right hemispheres in an individual One important difference between our method and the one brain is different from that in the template, this can lead to an employed in previous studies (Good et al., 2001; Watkins et al., inaccurate match, in which the brain gets slightly shifted to 2001) concerns the spatial preprocessing. In these studies, the one side, which can in turn cause asymmetrical spatially

948 DTI of White Matter Asymmetry • Büchel et al. normalized FA maps. To avoid these artifacts, we added an difficult to assess where precisely the difference is located and additional processing step in which we renormalized indi- how it relates to cortical areas. Thus further high resolution vidual hemispheres to a single average hemisphere, thus studies focusing on this area are necessary. allowing for individual normalization parameters for each hemisphere. Pilot analyses without this additional step Asymmetry Related to Handedness revealed asymmetry close to the midline in the left anterior and Using MR morphometry the only morphological difference right posterior part of the cingulate, that vanished after intro- related to handedness in the motor system is a deeper central ducing the additional single hemisphere normalization. Such sulcus in the hemisphere contralateral to the dominant hand an asymmetry close to the midline is to be treated with caution (Amunts et al., 2000). Interestingly this finding was obtained in because this implies abrupt changes in density of the corpus male subjects only. In contrast the difference in FA related to callosum, which seems biologically implausible (Watkins et al., handedness in our study was not significantly influenced by 2001). gender (data not shown). By smoothing segmented grey matter partitions, volume or Previous VBM studies failed to demonstrate any handedness- thickness differences can be transformed into image intensity related asymmetry in the human brain. Good and colleagues differences through the partial volume effect. These intensity reported asymmetry, but no effect of handedness, using VBM differences are then compared with a voxel-based statistic. on grey and white matter segmented T1-weighted images in Obviously, this works best if the smoothing kernel is large rela- 465 volunteers (Good et al., 2001). Another study used multi- tive to the structures of interest. In case of the thin gray matter modal imaging in 142 volunteers to better delineate the grey sheet, smoothing kernels of 12 mm have been used (Watkins et matter segment and performed voxel-based analyses on these al., 2001). Subcortical white matter structures are much segments (Watkins et al., 2001). Again, no consistent effect of thicker and therefore larger smoothing kernels are necessary. handedness was found. Both studies were based on sample This will, however, decrease the spatial resolution of the sizes much larger than ours. Hence, deficient statistical power analysis. is an unlikely reason for the null results obtained in these studies. General Asymmetry Recent DTI studies investigated the connectivity originating The left arcuate fascicule has been linked to language function from primary motor cortex in both hemispheres (Ciccarelli et in patients with conduction aphasia, which is characterized by al., 2003; Guye et al., 2003). In these studies DTI data were a predominant deficit in repetition (Geschwind, 1965) and is used in combination with fiber tracking algorithms to define often caused by a disruption of the arcuate fascicle, which macroscopic fiber tracts. One of these studies (Guye et al., connects temporal and frontal language regions. 2003) showed a more extensive connectivity in the dominant Our observation of greater fractional anisotropy in the left (left) hemisphere compared to the right hemisphere. The arcuate fascicle is also interesting with regard to studies of opposite comparison was not performed, because only right normal and abnormal white matter development and is in handers were investigated (Guye et al., 2003). In the other accord with a recent DTI region-of-interest-study showing study, no handedness-related asymmetry was found (Ciccarelli higher anisotropy in the white matter underneath the left et al., 2003). insula compared to the right insula (Cao et al., 2003). Using Our study is the first voxel-based analysis to reveal handed-

VBM on white matter segments from T1-weighted images, an ness-related differences. The important difference between our increase of white matter density was observed in the left study and previous studies is the imaging modality, i.e. diffu- arcuate fascicle only (Paus et al., 1999). This finding was sion-weighted imaging versus T1-weighted imaging. Our data further supported by a recent developmental DTI study strongly suggest that DTI is the imaging modality of choice for showing a correlation between FA and age in the left arcuate the investigation of white matter differences between groups fascicle (Schmithorst et al., 2002). and hemispheres. In addition, another study demonstrated that language Fractional anisotropy does not reflect a unique specific tissue impaired patients with dyslexia show decreased fractional property. Rather, FA is influenced by tissue hydration, myelina- anisotropy in this region (Klingberg et al., 2000). This decrease tion, cell-packing density and fiber diameter, and directional in FA was tightly correlated with reading performance. The coherence (Shimony et al., 1999; Virta et al., 1999). We are area of higher FA in the left hemisphere, as demonstrated in thus not in a position to uncover the exact nature of ultrastruc- our study, almost perfectly coincides with the observed tural changes in the precentral gyrus that seem to be associated decrease in FA in dyslexics. Assuming that the asymmetry in with handedness. the arcuate fascicle develops during language acquisition, one Comparing the location of the observed handedness-related could speculate that reduced FA in the left arcuate fascicle in FA with a probability map of Brodmann area (BA) 4 and the dyslexics is a sign of a deficient lateralization. pyramidal tract (Rademacher et al., 2001), shows that the coor- An asymmetry with greater FA values in the right hemisphere dinate falls precisely within the pyramidal tract. This suggests was found in the white matter of the inferior parietal lobe. that the difference observed is indeed located in the white Observations of neglect after right inferior parietal lesions that matter underneath BA 4. include the angular gyrus indicate a specific role of the right Microscopic studies of Brodmann area 4 revealed that the hemisphere in spatial processing (Mort et al., 2003). Hence, it hemisphere contralateral to the dominant hand shows is possible that the observed asymmetry reflects an asymmetry increased contents of neuropil (Amunts et al., 1996). The term in the neural structures of spatial processing. Although this ‘neuropil’ denotes a tissue compartment containing dendrites finding is robust as indicated by a replication in a second and axons. Although this relates mainly to tissue within the group, we refrain from interpreting the difference any further, boundary of grey matter, it is conceivable that remote effects because even in the analysis with a small smoothing kernel it is of increased neuropil can also be observed underneath the

Cerebral Cortex September 2004, V 14 N 9 949 grey matter sheet. This would suggest that increased FA in the Friston KJ, Holmes AP, Price CJ, Buchel C, Worsley KJ (1999) Multi- precentral gyrus is the result of a higher cell packing density subject fMRI studies and conjunction analyses. Neuroimage and more coherent fiber orientation. 10:385–396. Geschwind N (1965) Disconnexion syndromes in animals and man. Using DTI and voxel-based statistical analyses we were able Brain 88:237–294. to demonstrate a difference between the left and right arcuate Good CD, Johnsrude I, Ashburner J, Henson RNA, Friston KJ, fascicle. Together with developmental studies and studies of Frackowiak RSJ (2001) Cerebral asymmetry and the effects of sex language impairment, this strongly suggests a link to the and handedness on brain structure: a voxel-based morphometric language abilities of this hemisphere. In addition, our study is analysis of 465 normal adult human brains. Neuroimage the first to demonstrate a handedness-related difference in the 14:685–700. precentral gyrus using voxel-based statistics. These findings Guye M, Parker GJM, Symms M, Boulby P, Wheeler-Kingshott CAM, were remarkably robust, even when investigating small groups Salek-Haddadi A, Barker GJ, Duncan JS (2003) Combined functional MRI and tractography to demonstrate the connectivity of subjects, which highlights the sensitivity of DTI for white of the human primary motor cortex in vivo. Neuroimage matter tissue composition. This high sensitivity renders the 19:1349–1360. voxel-based analysis of DTI data an ideal tool to study patient Jäncke L, Schlaug G, Huang Y, Steinmetz H (1994) Asymmetry of the populations (i) in which an involvement of white matter tracts planum parietale. Neuroreport 5:1161–1163. is expected and (ii) in which a statement needs to be made Klingberg T, Hedehus M, Temple E, Salz T, Gabrieli JD, Moseley ME, with a small number of subjects. Recent examples of focal Poldrack RA (2000) Microstructure of temporo-parietal white differences in FA in stuttering (Sommer et al., 2002) and in matter as a basis for reading ability: evidence from diffusion tensor magnetic resonance imaging. Neuron 25:493–500. developmental dyslexia (Klingberg et al., 2000) underline this Koch MA, Glauche V, Finsterbusch J, Nolte UG, Frahm J, Weiller C, claim. Büchel C (2002) Distortion-free diffusion tensor imaging of cranial nerves and of inferior temporal and orbitofrontal white matter. Neuroimage 17:497–506. Supplementary Material Maguire EA, Gadian DG, Johnsrude IS, Good CD, Ashburner J, Supplementary material can be found at: http://www.cercor.oupjour- Frackowiak RS, Frith CD (2000) Navigation-related structural nals.org/ change in the hippocampi of taxi drivers. Proc Natl Acad Sci USA 97:4398–4403. May A, Ashburner J, Büchel C, McGonigle DJ, Friston KJ, Frackowiak Notes RS, Goadsby PJ (1999) Correlation between structural and func- We thank the Physics and Methods group of NeuroImage Nord in tional changes in brain in an idiopathic headache syndrome. Nat Hamburg for their support with scanning and A. Castro-Caldas, H. Med 5:836–838. Steinmetz and L. Jäncke for stimulating discussions. This work was Mort DJ, Malhotra P, Mannan SK, Rorden C, Pambakian A, Kennard C, supported by the Volkswagenstiftung (C.B.), a grant from the Univer- Husain M (2003) The anatomy of visual neglect. Brain sity of Göttingen (M.S.), the DFG and BMBF. 126:1986–1997. Address correspondence to Christian Büchel, NeuroImage Nord, Nolte UG, Finsterbusch J, Frahm J (2000) Rapid isotropic diffusion Haus S10, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, D- mapping without susceptibility artifacts: whole brain studies using 20246 Hamburg, Germany. Email: [email protected]. diffusion-weighted single-shot STEAM MR imaging. Magn Reson Med 44:731–736. Oldfield RC (1971) The assessment and analysis of handedness: the References Edinburgh inventory. Neuropsychologia 9:97–113. Amunts K, Schlaug G, Schleicher A, Steinmetz H, Dabringhaus A, Pajevic S, Pierpaoli C (1999) Color schemes to represent the orienta- Roland PE, Zilles K (1996) Asymmetry in the human motor cortex tion of anisotropic tissues from diffusion tensor data: application and handedness. Neuroimage 4:216–222. to white matter fiber tract mapping in the human brain. Magn Amunts K, Jancke L, Mohlberg H, Steinmetz H, Zilles K (2000) Inter- Reson Med 42:526–540. hemispheric asymmetry of the human motor cortex related to Paus T, Zijdenbos A, Worsley K, Collins DL, Blumenthal J, Giedd JN, handedness and gender. Neuropsychologia 38:304–312. Rapoport JL, Evans AC (1999) Structural maturation of neural path- Ashburner J, Friston KJ (2000) Voxel-based morphometry — the ways in children and adolescents: in vivo study. Science methods. Neuroimage 11:805–821. 283:1908–1911. Baron JC, Chetelat G, Desgranges B, Perchey G, Landeau B, de la Rademacher J, Burgel U, Geyer S, Schormann T, Schleicher A, Freund Sayette V, Eustache F (2001) In vivo mapping of gray matter loss HJ, Zilles K (2001) Variability and asymmetry in the human precen- with voxel-based morphometry in mild Alzheimer’s disease. tral motor system. A cytoarchitectonic and myeloarchitectonic Neuroimage 14:298–309. brain mapping study. Brain 124:2232–2258. Basser PJ (1995) Inferring microstructural features and the physiolog- Schmithorst VJ, Wilke M, Dardzinski BJ, Holland SK (2002) Correlation ical state of tissues from diffusion-weighted images. NMR Biomed of white matter diffusivity and anisotropy with age during child- 8:333–344. hood and adolescence: a cross-sectional diffusion-tensor MR Basser PJ, Mattiello J, LeBihan D (1994) Estimation of the effective self- imaging study. Radiology 222:212–218. diffusion tensor from the NMR spin echo. J Magn Resonon B Shimony JS, McKinstry RC, Akbudak E, Aronovitz JA, Snyder AZ, Lori 103:247–254. NF, Cull TS, Conturo TE (1999) Quantitative diffusion-tensor Cao Y, Whalen S, Huang J, Berger KL, DeLano MC (2003) Asymmetry anisotropy brain MR imaging: normative human data and anatomic of subinsular anisotropy by in vivo diffusion tensor imaging. Hum analysis. Radiology 212:770–784. Brain Mapp 20:82–90. Sommer M, Koch MA, Paulus W, Weiller C, Büchel C (2002) A discon- Ciccarelli O, Toosy AT, Parker GJM, Wheeler-Kingshott CAM, Barker nection of speech-relevant brain areas in developmental stuttering. 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950 DTI of White Matter Asymmetry • Büchel et al. pyramidal tract using diffusion tensor MRI. Magn Reson Imaging human brain: a voxel based statistical analysis of 142 MRI scans. 17:1121–1133. Cereb Cortex 11:868–877. Watkins KE, Paus T, Lerch JP, Zijdenbos A, Collins DL, Neelin P, Taylor Yousry TA, Schmid UD, Alkadhi H, Schmidt D, Peraud A, Buettner A, J, Worsley KJ, Evans AC (2001) Structural asymmetries in the Winkler P (1997) Localization of the motor hand area to a knob on the precentral gyrus. A new landmark. Brain 120:141–157.

Cerebral Cortex September 2004, V 14 N 9 951 MECHANISMS OF DISEASE

Mechanisms of disease

Disconnection of speech-relevant brain areas in persistent developmental stuttering

Martin Sommer, Martin A Koch, Walter Paulus, Cornelius Weiller, Christian Büchel

Summary Introduction Generation of fluent speech is dependent on the precise Background The neuronal basis of persistent developmental temporal synchronisation of phonatory and articulatory stuttering is unknown. The disorder could be related to a muscle groups.1 Language content modulates this process reduced left hemisphere dominance, which functional in a top-down fashion,2 indicating the close interaction neuroimaging data suggest might lead to right hemispheric between speech and language. This complex system is motor and premotor overactivation. Alternatively, the core severely compromised in developmental stuttering, a deficit underlying stuttering might be located in the speech- disorder that presents with involuntary repetitions, dominant left hemisphere. Furthermore, magnetoencephalo- lengthened sounds, or arrests of sounds and occurs in graphy study results show profound timing disturbances about 4–5% of all children3 aged 3–5 years. Although between areas involved in language preparation and stuttering can be characterised as a speech disorder, execution in the left hemisphere, suggesting that persistent symptoms seem specifically related to use of language, developmental stuttering might be related to impaired and are especially prominent in emotionally and neuronal communication, possibly caused by a disruption of syntactically demanding speech.2 Spontaneous remission white matter fibre tracts. We aimed to establish whether is common, but speech impairment persists after puberty disconnection between speech-related cortical areas was the in about 1% of people, more often in men than in structural basis of persistent developmental stuttering. women,3 and has a genetic basis.4 Despite decades of research, the origin of persistent developmental stuttering Methods We analysed the speech of 15 people with and its structural basis are unclear.2 persistent developmental stuttering and 15 closely matched Various theories of the pathophysiology underlying controls for the percentage of syllables stuttered. We used persistent developmental stuttering have been proposed: diffusion tensor imaging to assess participants’ brain tissue incomplete or abnormal patterns of cerebral hemispheric structure, and used voxel-based morphometry and two- dominance5,6 with a shift of activation to the right sample t test to compare diffusion characteristics between hemisphere, supported by experimental data for motor, groups. premotor,7,8 and auditory cortices;9 impaired oral motor control,10 with patients with persistent developmental Findings Diffusion characteristics of the group with stuttering showing slower reaction times in tasks persistent developmental stuttering and controls differed involving increasing complexity of bimanual decisions; 11 significantly immediately below the laryngeal and tongue impaired auditory self-monitoring of speech, supported representation in the left sensorimotor cortex (mean by temporal deactivation7,8,12,13 and negative correlation difference in fractional anisotropy 0·04 [95% CI 0·03–0·05]). between temporal activation and stuttering in positron emission tomography studies;8,12 and Interpretation Our findings show that persistent synchronisation deficits in speech preparation and developmental stuttering results from disturbed timing of execution, shown in a magnetoencephalography study.14 activation in speech-relevant brain areas, and suggest that The magnetoencephalography study results14 point right hemisphere overactivation merely reflects a towards disconnection of speech-related cortical areas in compensatory mechanism, analogous to right hemisphere the left hemisphere, which affects the synchronisation of activation in aphasia. motor and premotor cortex. We tested the hypothesis of a disconnection between Lancet 2002; 360: 380–83 speech-related cortical areas as the structural basis of persistent developmental stuttering by characterising brain tissue structure through DIFFUSION TENSOR IMAGING (DTI) in patients with persistent developmental stuttering. DTI can be used to measure the diffusion characteristics of water in vivo. The orientation of white matter fibre can be established with DTI because water diffuses faster if moving parallel rather than NeuroImage Nord, Department of Neurology, University of perpendicular to the longitudinal axis of axons.15 Hamburg, Hamburg, Germany (M Sommer MD, M A Koch PhD, FRACTIONAL ANISOTROPY (FA) OF DIFFUSION is a measure of Prof C Weiller MD, C Büchel MD); and Department of Clinical the coherence of the orientation of fibres within each Neurophysiology, University of Göttingen, Göttingen, Germany voxel (the smallest distinguishable box-shaped part of a (M Sommer, Prof W Paulus MD) three-dimensional space). In multiple sclerosis, lower FA Correspondence to: Dr C Büchel, NeuroImage Nord, Department of values can indicate decreased fibre coherence or Neurology, University of Hamburg, Martinistr 52, 20246 Hamburg, myelination defects.16 DTI has also been shown to be Germany sensitive to subtle white matter abnormalities in dyslexia, (e-mail: [email protected]) a developmental language disorder.17

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For personal use. Only reproduce with permission from The Lancet Publishing Group. MECHANISMS OF DISEASE

The full protocol consisted of a T2-weighted image and GLOSSARY six diffusion-weighted images sensitised for diffusion along six different directions. These measurements were repeated DIFFUSION TENSOR IMAGING (DTI) 40 times to improve the signal-to-noise ratio in the tensor An MRI technique sensitive to diffusion properties (direction, coherence) maps. A T1-weighted image was acquired by use of a three- of water protons. A set of six scans is obtained, each of which is dimensional fast-low-angle-shot sequence (flip angle 30º, sensitised for one of six non-colinear directions. TR 15 ms, TE 5 ms, 256256196 matrix, voxel size FRACTIONAL ANISOTROPY (FA) OF DIFFUSION 111 mm3). The first DTI image was discarded to A measure of the coherence of diffusion within each voxel. In highly exclude the transition to steady state. Image processing was ordered fibre bundles such as the corpus callosum, diffusion is mainly in done with SPM 99. DTI images were realigned with the the direction of, rather than perpendicular to, the fibre, resulting in high second image without diffusion weighting, and co- FA values. Low FA values can indicate decreased fibre coherence or myelination defects as seen in multiple sclerosis. registered with the high resolution T1 image, which we spatially normalised to a standard template,21 reorienting GAUSSIAN KERNEL the diffusion gradient directions accordingly. A three-dimensional, Gauss curve, used to filter images spatially The DTI images were sinc interpolated to 333 mm3 (ie, blur). The wider the Gaussian curve, the more blurring will result. resolution. The diffusion tensor and FA were established VOXEL-BASED MORPHOMETRY for every voxel.15 We compared FA between the fluent and An objective method to compare brain parenchyma between groups of stuttering groups using VOXEL BASED MORPHOMETRY. patients in each voxel of structural magnetic resonance images. Corrections for eddy current distortions were not necessary for this STEAM DTI acquisition. The FA maps were smoothed with a GAUSSIAN KERNEL of 6 mm full width at Methods half maximum. We studied 15 adults (five women) with persistent Because of the densely packed fibres, FA is higher in developmental stuttering (study group), no signs of white matter than in grey matter. Hence, FA could be cluttering, mean age 30·6 years (SD 7·5, range 18–44), and artificially higher in controls if the voxel of interest lay mean years of education 15·7 years (3·0, 10–20). The study predominantly in grey matter in the study group and in group came from all parts of Germany: 13 were registered white matter in controls. To keep this source of error to a with an experienced speech and language pathologist in minimum, we did a post-hoc analysis in which we Hamburg, who confirmed the diagnosis of persistent investigated the unsmoothed FA maps and compared the developmental stuttering; and two people had stuttering region of interest (101010 mm3 centred on the diagnosed by a neurologist with experience in speech- maximum [–48, –15, 18 mm]), between groups. Within language disorders (MS). this region of interest we established all voxels that We matched 15 controls for age (mean 30·0 years, contained white matter by use of the segmented high

SD 7·2, range 23–43), sex (four women), and years of resolution T1-weighted scan. Co-registration between education (mean 17·2 years, SD 2·7, range 15–23). diffusion weighted and T1-weighted images ensured a Controls had no personal or family history of stuttering or perfect match of structures between modalities. By contrast cluttering, were from the Hamburg region, and most were with echo planar diffusion weighted imaging, STEAM employees of the University of Hamburg hospital. Data for imaging does not introduce geometrical distortions due to one control could not be used (technical equipment susceptibility gradients or eddy currents. failure), which left 14 controls included in the study. For a statistical power of 0·8 and of 0·05, the sample No participants had a neurological or unstable medical size was sufficient to detect differences between stuttering disorder or took any drugs that acted on the central nervous patients and controls greater than 1 SD. To approximate a system. All participants apart from one stuttering patient normal distribution, behavioural data were log transformed: were right-handed with a score of at least 15 of 22 points on f(x)=ln(x+0·1), x=percentage syllables stuttered. Adding a the Edinburgh inventory.18 We obtained the agreement of constant was necessary to avoid a logarithm of 0 in one the Ethics Committee of the Medical Faculty of the Georg- control. FA values were compared between groups by use August-University of Göttingen, and written informed consent from all participants. 6 Immediately before doing DTI, we assessed the severity of stuttering (ie, repetitions, lengthened sounds, overt 5 speech blocks). Stuttering patients were asked to read aloud a newspaper article of 141 words and to talk spontaneously 4 about their perception of an international event. In the fluent control group, the text passage was longer (1273 3 words) to accurately score subtle speech abnormalities. Participants’ speech was recorded on audiotape and the 2 percentage of syllables stuttered assessed by two independent analysts.19 Individual percentages of syllables 1 stuttered were averaged across both analysts. DTI was done with a Magnetom Vision 1·5 T MR 0 system (Siemens; Erlangen, Germany) with a circularly Mean syllables stuttered (%) polarised head coil. Cushions were used to restrict –1 participants’ head movements. Participants wore earplugs for noise protection. We acquired DTI images with a –2 stimulated echo acquisition mode sequence (STEAM:20 flip Controls Study group angle 15º, TR [repetition time] 8872 ms, TE [echo time] 65 ms, 5664 matrix, field of view 168192 mm, and Figure 1: Percentage of syllables stuttered by the study group voxel size 335 mm3) of 20 slices covering the whole and controls brain apart from the cerebellum. Bars=95% CIs.

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For personal use. Only reproduce with permission from The Lancet Publishing Group. MECHANISMS OF DISEASE of SPM 99 (two-sample t test) with the threshold set to (figure 2). This region of reduced FA encompasses the p<0·05, corrected for multiple comparisons. white matter immediately below the sensorimotor representation of the oropharynx at the level of the central Role of the funding source sulcus (Brodmann’s area 43),8,12 close to variations in gyral The sponsors of the study had no role in the study design, anatomy reported in persistent developmental stuttering.22 data collection, data analysis, data interpretation, writing Post-hoc analysis of the region of interest showed of the report, or decision to submit the paper for a significant difference between groups (mean difference publication. in FA 0·03 [95% CI 0·01–0·05]; mean of relative FA reduction in study group 15·1% [4·6–25·6], uncorrected Results p=0·0036), indicating FA differences within white matter Patients in the study group stuttered significantly more (figure 2). The lower significance level in the post-hoc syllables (mean % stuttered syllables 4·04% [95% CI analysis was expected because the analysis was based on 2·42–5·66], range 1·0–20·4), than controls (0·12% [–1·17 unsmoothed FA data to avoid further grey-white matter to 1·41], range 0–0·9, one-tailed unpaired t test smearing. p=1·4810–10; figure 1). The interanalyst reliability of scores was 0·85. Discussion FA was significantly lower in the study group than Our results show signs of a cortical disconnection in controls (mean difference 0·04 [95% CI 0·03–0·05]; people with persistent developmental stuttering mean of relative FA reduction 32·8% [22·3–43·3], immediately below the laryngeal and tongue p=0·014, corrected for multiple comparisons in the whole representation in left sensorimotor cortex. The immediate brain volume), in the rolandic operculum of only the surrounding region is composed of the sensorimotor left hemisphere, immediately above the Sylvian fissure representation of tongue, larynx, and pharynx in the subcentral sulcus; the ventral extension of the central 23 A Central sulcus sulcus; and the inferior arcuate fascicle linking temporal and frontal language areas.24,25 In particular, fibre tracts in this area connect the sensorimotor representation of the oropharynx with the frontal operculum involved in articulation26 and the ventral premotor cortex related to the planning of motor aspects of speech.25 Thus, disturbed signal transmission through the left rolandic operculum could impair sensorimotor integration necessary for fluent speech production.1,27 Our findings show that the normal temporal pattern of activation in premotor and motor cortex is disturbed14 and, as a consequence, that right hemisphere language areas compensate for this deficit,7,8 similar to recovery in aphasia.28 The alternative interpretation, that right hemisphere overactivation is the cause of persistent developmental stuttering and leads to subsequent atrophy of white matter in the left rolandic operculum as a secondary effect, cannot be discounted by our data. However, this Rolandic operculum mechanism is extremely unlikely: it is unclear how widespread right hemisphere overactivation could lead to B a focal abnormality in the left hemisphere; and right 0·22 hemisphere abnormality does not provide a plausible explanation for the synchronisation abnormality in the left 14 0·21 hemisphere noted with magnetoencephalography. Furthermore, a possible disconnection, as shown by our data, is consistent with decreased activation of the 0·20 adjacent posterior inferior frontal cortex during stuttering.7,29 0·19 Fluency inducing techniques such as chorus reading or shadowing have a powerful effect in stuttering.2 Chorus reading and shadowing might induce fluency by providing 0·18 an external clock. Through projections from periauditory Fractional anisotropy areas, this external clock might be able to functionally 0·17 compensate the disconnection between frontal speech planning areas and motor areas by synchronising their activity via a common input. In accord with this theory, 0·16 fluency-inducing procedures such as chorus reading Study group Controls increase activity in temporal areas.7 Figure 2: Voxels with significantly lower FA in the study group We can only speculate about the histological alterations than in controls (top panel), and comparison of post-hoc FA underlying the difference in FA. Although brain analysis results (bottom panel) myelination is essentially complete at age 5 years,30 results Voxels are superimposed in red on a normalised T1-weighted anatomical from DTI studies in children aged 5–17 years31 showed an image of a control at a threshold of p<0·001 (size of cluster=81 mm3). The insert is cut at x<–48 mm, y between –40 and 5 mm, and z>18 mm; increase of FA with age, indicating that DTI is a sensitive coordinates refer to the space defined by the Montreal Neurological marker for white matter development beyond Institute. Bars in the lower panel=SEs. myelination. Similarly, results of a morphometric study

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21 using T1-weighted imaging showed a correlation of white 8 Braun AR, Varga M, Stager S, et al. Altered patterns of cerebral activity during speech and language production in developmental matter density in T1-weighted images with age. In both studies, the correlation was most prominent in the motor stuttering: an H2(15)O positron emission tomography study. Brain 1997; 120: 761–84. system (internal capsule) and the speech-language system 9 Salmelin R, Schnitzler A, Schmitz F, Jancke L, Witte OW, Freund (left frontoparietal white matter). This finding parallels HJ. Functional organisation of the auditory cortex is different in the development of these functional systems during stutterers and fluent speakers. Neuroreport 1998; 9: 2225–29. childhood and adolescence. Since the density of white 10 DeNil LF. Stuttering: bridging the gap between theory and practice. matter obtained with T and FA show a similar correlation In: Bernstein Ratner N, Healey EC, eds. Hillsdale: Erlbaum, 1999: 1 85–102. with age, the increase in FA after the completion of 11 Webster WG, Ryan CR. Task complexity and manual reaction times myelination probably arises from an increase in density in people who stutter. J Speech Hear Res 1991; 34: 708–14. and coherence of neuronal packing.31 12 Fox PT, Ingham RJ, Ingham JC, Zamarripa F, Xiong JH, A limitation of our study follows from the large voxel Lancaster JL. Brain correlates of stuttering and syllable production: size that we chose because of little a-priori knowledge. a PET performance-correlation analysis. Brain 2000; 123: Therefore, we do not know whether the FA difference is 1985–2004. 13 Ingham RJ, Fox PT, Costello Ingham J, Zamarripa F. Is only in white matter or whether the grey-white border is overt stuttered speech a prerequisite for the neural activations also involved. A further limitation is that we did not associated with chronic developmental stuttering? Brain Lang 2000; control for any past or present therapy in the study group, 75: 163–94. which could have improved fluency scores and modified 14 Salmelin R, Schnitzler A, Schmitz F, Freund HJ. Single word reading correlation between FA scores and fluency. The small in developmental stutterers and fluent speakers. Brain 2000; 123: 1184–202. sample size restricts our study only with regard to 15 Basser PJ, Mattiello J, LeBihan D. Estimation of the effective self- detection of strong differences of the mean between diffusion tensor from the NMR spin echo. J Magn Reson B 1994; groups. 103: 247–54. Overall, our findings provide evidence for a structural 16 Filippi M, Cercignani M, Inglese M, Horsfield MA, Comi G. abnormality in speech-relevant areas in the left rolandic Diffusion tensor magnetic resonance imaging in multiple sclerosis. Neurology 2001; 56: 304–11. operculum in persistent developmental stuttering. This 17 Klingberg T, Hedehus M, Temple E, et al. Microstructure of abnormality probably develops during the period of early temporo-parietal white matter as a basis for reading ability: evidence language and speech acquisition in which many children from diffusion tensor magnetic resonance imaging. Neuron 2000; 25: experience a transient phase of stuttering.2 Our methods 493–500. could be used to ascertain why certain children develop 18 Oldfield RC. The assessment and analysis of handedness: the persistent stuttering whereas others become fluent Edinburgh inventory. Neuropsychologia 1971; 9: 97–113. 19 Onslow M, Costa L, Andrews C, Harrison E, Packman A. Speech speakers. outcomes of a prolonged-speech treatment for stuttering. J Speech Hear Res 1996; 39: 734–49. Contributors 20 Nolte UG, Finsterbusch J, Frahm J. Rapid isotropic diffusion M Sommer was involved in planning, study coordination, manuscript mapping without susceptibility artifacts: whole brain studies using writing, and data analysis. M Koch was involved in implementing MR diffusion-weighted single-shot STEAM MR imaging. pulse sequences and data analysis. W Paulus and C Weiller were involved Magn Reson Med 2000; 44: 731–36. in acquisition of funding and in planning the study. C Büchel was 21 Paus T, Zijdenbos A, Worsley K, et al. Structural maturation of involved in study design, data analysis, review of data, manuscript writing, neural pathways in children and adolescents: in vivo study. Science and overall study coordination. 1999; 283: 1908–11. 22 Foundas AL, Bollich AM, Corey DM, Hurley M, Conflict of interest statement Heilman KM. Anomalous anatomy of speech-language areas in None declared. adults with persistent developmental stuttering. Neurology 2001; 57: 207–15. Acknowledgments 23 Wildgruber D, Ackermann H, Klose U, Kardatzki B, Grodd W. This work was supported by Volkswagenstiftung (C Büchel, M Koch), the Functional lateralization of speech production at primary motor European Commission (C Weiller; 5th framework), and a grant from the cortex: a fMRI study. Neuroreport 1996; 7: 2791–95. Medical Faculty of Göttingen (M Sommer). We thank A Starke for the 24 Paulesu E, Frith CD, Frackowiak RS. The neural correlates of assessment of persistent developmental stuttering patients; the Department the verbal component of working memory. Nature 1993; 362: of Neurology, the radiographers, and information technology team of the 342–45. Cognitive Neuroscience Laboratory for help with scanning; 25 Price CJ, Wise RJ, Warburton EA, et al. Hearing and saying: the J Finsterbusch and J Frahm for providing the turbo STEAM DTI sequence; functional neuro-anatomy of auditory word processing. Brain 1996; M Sach, V Glauche, and R Knab for help with data analysis; and 119: 919–31. O Tüscher and T Wolbers for suggestions on an earlier draft of this paper. 26 Wise RJ, Greene J, Buchel C, Scott SK. Brain regions involved in articulation. Lancet 1999; 353: 1057–61. 27 Tonkonogy J, Goodglass H. Language function, foot of the third References frontal gyrus, and rolandic operculum. Arch Neurol 1981; 38: 1 Perkins WH, Kent RD, Curlee RF. A theory of neuropsycholinguistic 486–90. function in stuttering. J Speech Hear Res 1991; 34: 734–52. 28 Weiller C, Isensee C, Rijntjes M, et al. Recovery from Wernicke’s 2 Bloodstein O. A handbook on stuttering. San Diego: aphasia—a positron emission tomographic study. Ann Neurol 1995; Singular Publishing Group, 1995. 37: 723–32. 3 Yairi E, Ambrose NG. Early childhood stuttering I: persistency 29 Wu JC, Maguire G, Riley G, et al. A positron emission tomography and recovery rates. J Speech Lang Hear Res 1999; 42: 1097–112. [18F]deoxyglucose study of developmental stuttering. Neuroreport 4 Ambrose NG, Cox NJ, Yairi E. The genetic basis of persistence 1995; 6: 501–05. and recovery in stuttering. J Speech Lang Hear Res 1997; 40: 567–80. 30 Nakagawa H, Iwasaki S, Kichikawa K, et al. Normal myelination of 5 Travis LE. The cerebral dominance theory of stuttering: 1931–1978. anatomic nerve fiber bundles: MR analysis. AJNR Am J Neuroradiol J Speech Hear Disord 1978; 43: 278–81. 1998; 19: 1129–36. 6 Orton ST. A physiological theory of reading disability and stuttering in 31 Schmithorst VJ, Wilke M, Dardzinski BJ, Holland SK. Correlation of children. N Engl J Med 1928; 199: 1046–52. white matter diffusivity and anisotropy with age during childhood 7 Fox PT, Ingham RJ, Ingham JC, et al. A PET study of the neural and adolescence: a cross-sectional diffusion-tensor MR imaging systems of stuttering. Nature 1996; 382: 158–61. study. Radiology 2002; 222: 212–18.

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Modi®cation of the silent period by double transcranial magnetic stimulation

Tao Wu, Martin Sommer, Frithjof Tergau, Walter Paulus*

Department of Clinical Neurophysiology, Georg August University, Robert-Koch-Street 40, D-37075 GoÈttingen, Germany Accepted 18 July 2000

Abstract Objectives: To study the time course of the changes of the inhibitory network of the human motor system, we investigated the silent period (SP) in 7 healthy subjects by double suprathreshold transcranial magnetic stimulation (TMS). Methods: SPs and motor evoked potentials (MEPs) were recorded from the voluntarily activated right abductor digiti minimi muscle. Conditioning and test stimuli were delivered with equal intensity, which was set to yield a baseline SP duration of 130 ms by a single pulse, and with various interstimulus intervals (ISIs). In addition, a control experiment with adjustment of the intensity of single stimuli was performed. Results: At ISIs of 20 and 30 ms the test pulse SP duration was prolonged, without increasing the MEP amplitude. The SP duration shortened at longer ISIs and showed a signi®cant depression between ISIs of 60±110 ms. The shortened SP was accompanied by a diminished MEP. The control experiment revealed that the SPs evoked by the adjusted pulses were signi®cantly shorter than the test pulse SPs. Conclusions: A conditioning stimulus can prolong and shorten the test pulse SP duration at different ISIs. The prolongation is probably cortically generated, whereas the shortening is likely to occur at a cortical and spinal level. q 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Silent period; Double transcranial magnetic stimulation; Motor cortex; Inhibitory system

1. Introduction (Cantello et al., 1992), the SP has a lower active threshold than the MEP (Wassermann et al., 1993) and the MEP Transcranial magnetic stimulation (TMS) of the motor amplitude plateaus while the SP duration continues to cortex during voluntary contraction produces a motor increase at a high stimulus intensity (Inghilleri et al., evoked potential (MEP) followed by a period of silence 1993). Several authors have described the procedures of (SP) in the background electromyographic (EMG) activity the changes of the MEP size and motor threshold after a (Rothwell et al., 1991). The origin of the SP has been conditioning cortical stimulation (Valls-SoleÂ et al., 1992; attributed to several mechanisms. Recent studies indicate Wassermann et al., 1996; Tergau et al., 1999). Although that although spinal mechanisms such as after-hyperpolar- these ®ndings re¯ected not only the activation of the exci- ization and Renshaw cell activation may contribute to the tatory, but also the inhibitory network, the latter was only ®rst part, the major portion of the SP is due to inhibitory indirectly probed. cortical mechanisms (Cantello et al., 1992; Uozumi et al., With the use of two suprathreshold pulses the in¯uence 1992; Inghilleri et al., 1993; Roick et al., 1993; Ziemann et of a ®rst conditioning stimulus on the motor system al., 1993; Hallett, 1995). could be detected through the motor response evoked by There is some evidence which suggests that the excita- the second test pulse (Valls-SoleÂ et al., 1992; Wassermann tory and inhibitory networks are activated differently by et al., 1996). In the present study, we employed this TMS: the SP can be recorded without a preceding MEP double-pulse paradigm to investigate the duration of the SP, which represents the inhibitory effect directly (Rossini et al., 1994), as the parameter to measure the time course of the changes of the activity of the inhibitory * Corresponding author. Tel.: 149-551-39-66-50; fax: 149-551-39-81- system. 26. E-mail address: [email protected] (W. Paulus).

1388-2457/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. CLINPH 99624 PII: S1388-2457(00)00426-0 T. Wu et al. / Clinical Neurophysiology 111 (2000) 1868±1872 1869 2. Materials and methods SP duration was performed at each intensity. The intensity used for the double stimuli was de®ned as the ®rst stimulus 2.1. Subjects intensity that yielded an average SP duration of 130 ms in 5 subsequent trials. Each experiment started with 10 single We investigated 7 healthy volunteers (4 male, 3 female; pulses at this intensity. We averaged these 10 SP durations average age 27.1 years, range 22±30 years). The experi- and MEP amplitudes as baseline of SP (bSP) and MEP ments were performed according to the Declaration of (bMEP), respectively. Subsequently, double pulses were Helsinki and were approved by the Ethics Committee of delivered with interstimulus intervals (ISIs) of 20±200 ms the Medical Faculty of the University of GoÈttingen. All in steps of 10 ms. Four blocks of trials were performed, each volunteers gave written informed consent to participate in consisting of 4 or 5 randomly intermixed ISIs (5 trials each). this study. The intertrial interval of all experiments was 5 s. After each block the subjects had a short rest of about 10 min. 2.2. Stimulation and recording technique

Subjects were seated in a comfortable reclining chair. 2.3. Statistical methods Surface EMG recordings were made from the right abductor digiti minimi muscle (ADM) with Ag/AgCl electrodes ®xed The duration of the SP evoked by a single pulse was in a belly-tendon montage. The raw signal was ampli®ed measured from the beginning of the MEP to the recovery and ®ltered (time constant 10 ms, low-pass ®lter 3.0 kHz, of voluntary EMG activity. For double stimuli, at short ISIs high-pass ®lter 5.0 Hz) and then fed into an IBM-PC/486 (,50 ms), the determination of the beginning of the SP AT-compatible laboratory computer, using the NEUROS- evoked by the test pulse was dif®cult as the stimulus artifact CAN (version 3.0) data collection (sampling rate 5 kHz) and and the MEP produced by the test pulse appeared before the conditional averaging software. Focal TMS was delivered to end of the MEP induced by the ®rst stimulus. We de®ned the the hand area of the left motor cortex through a ®gure-of- duration of the test pulse SPs from the onset of the ®rst MEP eight coil (outer diameter of one winding 70 mm) connected to the recovery of voluntary EMG activity after the test to two Magstim 200 magnetic stimulators via a BiStim pulse, then subtracted the corresponding ISI of each trial module (maximum output 2.0 T, The Magstim Company, (Fig. 1). The duration of the SP and the amplitude of the Whitland, Dyfed, UK). The optimal position for activation MEP induced by the test pulse were expressed as a percen- of the ADM was found by moving the coil in 1 cm steps tage of the bSP and bMEP, respectively. Student's t test was around the presumed area of the left motor cortex and was used to analyze the differences of the SP or MEP from the de®ned as the site where stimuli of moderately suprathres- baseline values at each ISI, while the values at ISIs of 20 and hold intensity consistently produced the largest MEPs in the 30 ms were also calculated by the Wilcoxon test. The rela- target muscle. The handle of the coil was held in a postero- tionship of the SPs and MEPs was detected with linear lateral direction so that the current induced in the brain ¯ow regression. For all experiments, data are given as the was perpendicular to the central sulcus. This position was marked with a pen in order to ensure a constant placement of the coil throughout the experiment. The resting motor threshold (RMT) was determined in the completely relaxed ADM under auditory EMG feedback control. The threshold intensity, expressed as a percentage of maximum stimulator output, was approached from suprathreshold levels by reducing the stimulus intensity in 1% steps and was de®ned as the ®rst stimulus intensity that failed to produce an MEP of more than 50 mV in 5 subsequent trials (Rossini et al., 1994). The effect of double-pulse TMS on the duration of the SP was studied in 25% maximum activated right ADM. The subjects' force of voluntary contraction was measured by a Fig. 1. Typical example of the silent period (SP) in the voluntary activated vigorimeter and controlled with visual feedback using an right ADM (25% of maximal contraction) after double magnetic stimuli oscilloscope monitor in front of them. The double stimuli with equal intensity of the contralateral motor cortex. The interstimulus were delivered with equal stimulus intensity and through the interval (ISI) is 30 ms. The arrows from left to right indicate: the artifact same coil. The ®rst and second stimuli are referred to as the of the ®rst (conditioning) pulse (A); the onset of the ®rst motor evoked conditioning and test stimuli, respectively. The intensity of potential (MEP) (B); the onset of the second (test) MEP (C); the recovery of EMG burst at the end of the SP (D). We measured the duration of the test the conditioning and test stimuli was determined by increas- pulse SP from the onset of the ®rst MEP to the end of the SP (arrow B to ing the intensity of a single stimulator from the motor arrow D), then subtracted the corresponding ISI (30 ms in this case). The threshold level in 1% steps. An on-line averaging of the test pulse SP duration is 217 ms. 1870 T. Wu et al. / Clinical Neurophysiology 111 (2000) 1868±1872 mean ^ standard error (SE), and statistical signi®cance was was 57.6 ^ 2.4% (range 47±65%) and was about 1.25 fold assumed if P , 0:05. of RMT (range 1.08±1.45 fold). The average duration of the bSP was 134.3 ^ 1.9 ms (range 125.4±137.8 ms). 2.4. Control experiment 1 3.2. Time course of the SPs evoked by test pulses To study the effect of the level of contraction on the change of the SPs and MEPs, we performed double-pulse The averaged normalized test pulse SP duration and the TMS in 10% activated right ADM in all 7 subjects. The MEP amplitudes for all 7 subjects at each ISI are shown in stimuli were delivered as in the main experiment. Data Fig. 2. At short ISIs (20±30 ms) all subjects showed a signif- analysis was identical to the principal experiment. We icant prolongation of the SP by either t test (P , 0:05) or used analysis of variance (ANOVA) to compare the results Wilcoxon test (P , 0:005). In one subject, this prolongation of this control session to those of the principal experiment. was present up to an ISI of 40 ms. The duration of the SP decreased gradually with increasing ISI and showed a 2.5. Control experiment 2 signi®cant shortening between ISIs of 60±110 ms. At an ISI of 160 ms the SP duration returned to baseline again, In order to further analyze the mechanisms responsible and an insigni®cant prolongation occurred at ISIs of for the change of the SPs, a control experiment was 160±200 ms. performed in 4 subjects. We applied 45 single pulses with 9 different intensities, which were adjusted to induce the MEP amplitudes from 10 to 90% of the bMEP, imitating 3.3. Comparison of the SPs and the MEPs the MEP amplitudes evoked by the test stimuli of double The time course of the MEPs showed nearly the same TMS at different ISIs. Five trials were recorded for each trend as that of the SPs (Fig. 2). However, at ISIs of 20 intensity with an interval of 5 s. SPs induced by these and 30 ms we did not ®nd a facilitation of the MEPs. At adjusted stimuli were measured and normalized to the an ISI of 40 ms the MEP began to decrease and was signi®- bSP. ANOVA was used to compare the values of the SP cantly inhibited from 40 to 140 ms. The MEP recovered evoked by adjusted stimuli to the SPs produced by test completely at an ISI of 170 ms. The result of linear regres- pulses of double stimuli. Data analysis was identical to sion between the time course of the SPs and MEPs (Fig. 3) the principal experiment. showed a high correlation (r 0:927).

3. Results 3.4. Results of the control experiment 1

3.1. RMT and stimulus intensity The results of the SPs and the MEPs at 10% contraction were nearly the same as those at 20% voluntary activation The average RMT in all 7 subjects was 46.0 ^ 3.1% (ANOVA, P . 0:05). At ISIs of 20 and 30 ms, a signi®- (range 40±60%). The average stimulus intensity of a single cantly prolonged SP duration appeared, without a facilita- pulse to achieve an average baseline SP duration of 130 ms tion of the MEPs. At ISIs ranging from 40 to 150 ms MEPs were reduced in size, and at ISIs from 60 to 120 ms the SP was shortened. There was not a signi®cant interaction of the level of contraction by parameter (SP, MEP) or of the level of contraction by ISI.

Fig. 2. The time course of the SPs and MEPs evoked by the test pulses during voluntary contraction of right ADM (SP, ®lled circles; MEP, open circles) at different ISIs. All results are normalized to the baseline SP (bSP) and MEP (bMEP), respectively (baseline set to 100%). The grand mean of 5 trials of each ISI in 7 subjects is shown with standard errors. A signi®cant Fig. 3. The result of linear regression is shown as the amplitude of test pulse difference to baseline (Student's t test) is indicated by *P , 0:05 or MEP (x-axis) against the duration of the test pulse SP (y-axis). **P , 0:01. y 59:869 1 0:501x; r 0:927, P , 0:0001. T. Wu et al. / Clinical Neurophysiology 111 (2000) 1868±1872 1871 3.5. Results of the adjusted SPs prolongation of SP; the latter is thus likely to be due to the increased excitability of the inhibitory system at a suprasp- The averaged values of the SPs evoked by the adjusted inal level. Our results are consistent with a recent report single pulses in 4 subjects were signi®cantly shorter than the (Shimizu et al., 1999) in which the authors also used paired SPs produced by the test pulses of the double stimuli (see TMS and found a prolongation of SP at ISIs of 15±20 ms Fig. 4) (ANOVA, effect of stimulus condition, P , 0:05), without alteration of MEP size. Similar to us, the authors particularly when adjusted with lower MEP amplitudes contribute the SP prolongation to supraspinal mechanisms. (from 10 to 40% of the bMEP; post-hoc, Fishers test, The origin of the cortical SP remains debatable, but inhi- P , 0:05). bitory postsynaptic potentials (IPSPs) produced by intracortical inhibitory GABA interneurons is a widely accepted explanation (Krnjevic et al., 1966a,b; Fuhr et al., 1991; 4. Discussion Hallett, 1995). Cortical stimulation can activate a horizontal network of intracortical inhibitory interneurons (Asanuma The principal ®nding of the present paper is that the silent and RoseÂ, 1973), which might induce a spread of IPSPs period is prolonged at short ISIs (,40 ms) and is shortened (Kang et al., 1994) and produce a cortical silent period. at long ISIs (.40 ms) by double suprathreshold TMS. Since Presumably, the spatial and temporal summation of IPSPs the ®rst and the second MEP may overlap at ISIs of less than evoked by the test pulse with that produced by the condi- 20 ms, we did not give double TMS at shorter intervals. tioning stimulus would prolong the cortical SP considerably. This explanation is supported by previous reports 4.1. The prolongation of the SP that double pulses in somatosensory neocortical or hippocampal slices were able to elicit enhanced IPSPs at short A suprathreshold conditioning stimulus signi®cantly ISIs (Nathan and Lambert, 1991; Fleidervish and Gutnick, prolongs the SP duration at ISIs of 20 and 30 ms. Although 1995). spinal mechanisms may operate in the ®rst 50 ms, the major part of the SP induced by cortical stimulation is attributed to cortical mechanisms (Fuhr et al., 1991; Uozumi et al., 1992; 4.2. The shortening of the SP Inghilleri et al., 1993; Roick et al., 1993; Ziemann et al., 1993; Hallett, 1995). As the MEP amplitude re¯ects the We attribute the shortening of the SP duration at long ISIs number of simultaneously activated spinal motoneurons, to either cortical or spinal inhibitory mechanisms. The the spinal SP duration is correlated to the preceding MEP depressed SP was consistently preceded by a diminished size (Triggs et al., 1993). In contrast, the cortical SP is MEP which indicates that such a change is partly due to independent from the MEP amplitude (Inghilleri et al., spinal mechanisms. The decreased motor response to the 1993). In our study, we did not ®nd a signi®cantly increased test pulse is produced by the reduced excitatory motor MEP size preceding the prolonged SP duration. This indi- drive on the cortico-muscular pathway after a conditioning cates that an enhanced spinal inhibition is not crucial for the stimulus (Valls-SoleÂ et al., 1992; Wassermann et al., 1996; Tergau et al., 1999). These descending volleys of lower amplitude should activate less spinal inhibitory networks and, therefore, induce a shortened SP. However, the shortened SP was not purely due to the altered activation of the spinal inhibition, as the SPs evoked by the adjusted single pulses were signi®cantly different from the test pulse SPs preceded by the same MEP amplitude (Fig. 4). Further support derives from the comparison of the test pulse SPs and MEPs in detail. For example, the shortest SP duration appeared at an ISI of 80 ms, in which the SP was about 60% of the bSP and was diminished for more than 50 ms. The corresponding test MEP size was 20% of the bMEP, which suggested that the spinal SP was still Fig. 4. SPs evoked by the test pulses of double TMS (®lled circles) or by single pulses (open squares) at different preceding MEP amplitudes. The existent (Fig. 2). These results indicate that the cortical SP is intensities of the single pulses were adjusted to yield MEPs that were in also shortened at the long ISI. We speculate that the activity amplitude from 10 to 90% of the bMEP, imitating the amplitudes evoked by of the intracortical inhibitory interneurons evoked by the the test pulses of double stimuli at different ISIs. The duration of the SPs conditioning pulse decreases after a relatively long time, and the amplitudes of MEPs are normalized to the bSP and bMEP, respec- and the quantal contents for IPSPs decay at this period, so tively (baseline set to 100%). The grand mean of 5 trials of each intensity in 4 subjects is shown with standard errors. A signi®cant difference between that the subsequent stimulus might result in much smaller the adjusted SPs and the test pulse SPs (post-hoc, Fisher's test) is indicated IPSPs (Wilcox and Dichter, 1994; Fleidervish and Gutnick, by *P , 0:05 or **P , 0:01. 1995). 1872 T. Wu et al. / Clinical Neurophysiology 111 (2000) 1868±1872

4.3. The lack of facilitation of the MEPs at short ISIs Hallett M. Transcranial magnetic stimulation. Negative effects. In: Fahn S, Hallett M, LuÈders HO, Marsden CD, editors. Negative motor phenom- Our results con®rm that double suprathreshold TMS does ena, Philadelphia, PA: Lippincott-Raven, 1995. pp. 107±113. not facilitate the MEP size at ISIs of 20±30 ms while the Inghilleri M, Berardelli A, Cruccu G, Manfredi M. Silent period evoked by subjects activate the target muscle (Wassermann et al., transcranial stimulation of the human cortex and cervicomedullary junction. J Physiol (Lond) 1993;466:521±534. 1996). This ®nding is also parallel to a previous study Kang Y, Kaneko T, Ohishi H, Endo K, Araki T. Spatiotemporally differ- (Tergau et al., 1999), which explored that AMT did not ential inhibition of pyramidal cells in the cat motor cortex. J Neurophy- change shortly after the conditioning stimulus. In contrast, siol 1994;71:280±293. an increased MEP size at short ISIs can be detected in a Krnjevic K, Randic M, Straughan DW. An inhibitory process in the cerebral resting muscle (Valls-SoleÂ et al., 1992; Nakamura et al., cortex. J Physiol (Lond) 1966a;184:16±48. Krnjevic K, Randic M, Straughan DW. Nature of a cortical inhibition 1997). A possible explanation for the phenomenon is that process. J Physiol (Lond) 1966b;184:49±77. the excitability of the spinal and cortical motoneurons is Nakamura H, Kitagawa H, Kawaguchi Y, Tsuji H. Intracortical facilitation increased by voluntary contraction, so that a given stimula- and inhibition after transcranial magnetic stimulation in conscious tion enhances the MEP size more than in the resting muscle humans. J Physiol (Lond) 1997;498:817±823. (Di Lazzaro et al., 1998). Therefore, less additional moto- Nathan T, Lambert JDC. Depression of the fast IPSP underlies paired-pulse neurons are recruited by the test pulse. However, 3 pieces of facilitation in area CA1 of the rat hippocampus. J Neurophysiol 1991;66:1704±1715. evidence suggest that the disfacilitated MEP is more likely Roick H, Giesen HJV, Benecke R. On the origin of the post-excitatory due to the activation of the inhibitory network. First, the SP inhibition seen after transcranial magnetic stimulation in awake duration at short ISIs is signi®cantly increased (Fig. 2). human subjects. Exp Brain Res 1993;94:489±498. Second, voluntary contraction can enhance the activation Rossini PM, Barker AT, Berardelli A, Caramia MD, Caruso G, Cracco RQ, of the inhibitory system (Triggs et al., 1993). Consequently, Dimitrijevic MR, Hallett M, Katayama Y, Lucking CH, Maertens de Noordhout AL, Marsden CD, Murray NMF, Rothwell JC, Swash M, the enhanced activity of the cortical inhibitory network Tomberg C. 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