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Synergistic effects of noradrenergic modulation with atomoxetine and 10�hz repetitive transcranial magnetic stimulation on motor learning in healthy humans

Sczesny-Kaiser et al. BMC Neuroscience 2014, 15:46http://www.biomedcentral.com/1471-2202/15/46 Synergistic effects of noradrenergic modulationwith atomoxetine and 10 Hz repetitivetranscranial magnetic stimulation on motorlearning in healthy humans Matthias Sczesny-Kaiser1*†, Alica Bauknecht1†, Oliver Höffken1, Martin Tegenthoff1, Hubert R Dinse2, Dirk Jancke2,Klaus Funke3 and Peter Schwenkreis1 Background: Repetitive transcranial magnetic stimulation (rTMS) is able to induce changes in neuronal activity thatoutlast stimulation. The underlying mechanisms are not completely understood. They might be analogous tolong-term potentiation or depression, as the duration of the effects seems to implicate changes in synapticplasticity. Norepinephrine (NE) has been shown to play a crucial role in neuronal plasticity in the healthy and injuredhuman brain. Atomoxetine (ATX) and other NE reuptake inhibitors have been shown to increase excitability indifferent systems and to influence learning processes. Thus, the combination of two facilitative interventions maylead to further increase in excitability and motor learning. But in some cases homeostatic metaplasticity mightprotect the brain from harmful hyperexcitability. In this study, the combination of 60 mg ATX and 10 Hz rTMS overthe primary motor cortex was used to examine changes in cortical excitability and motor learning and toinvestigate their influence on synaptic plasticity mechanisms.
Results: The results of this double-blind placebo-controlled study showed that ATX facilitated corticospinal andintracortical excitability in motor cortex. 10 Hertz rTMS applied during a motor task was able to further increaseintracortical excitability only in combination with ATX. In addition, only the combination of 10 Hz rTMS and ATXwas capable of enhancing the total number of correct responses and reaction time significantly, indicating aninteraction effect between rTMS and ATX without signs of homeostatic metaplasticity.
Conclusion: These results suggest that pharmacologically enhanced NE transmission and 10 Hz rTMS exert asynergistic effect on motor cortex excitability and motor learning in healthy humans.
Keywords: rTMS, Neuromodulation, Norepinephrine, Atomoxetine, Plasticity, Motor cortex different TMS-protocols. Furthermore, rTMS is capable Repetitive transcranial magnetic stimulation (rTMS) is a to influence task performance and learning processes. For non-invasive tool for brain stimulation and is able to example, it can improve motor learning The induced modulate brain activity beyond stimulation The effects depends on different parameters like coil orientation, mechanisms underlying these long-term rTMS-effects total number of pulses and frequency.
could be analogous to long-term potentiation (LTP) or For the measurement of rTMS-induced changes of depression (LTD). These rTMS-induced changes in cor- cortical excitability, a single-pulse TMS (spTMS) protocol tical excitability and brain activity can be measured by called "stimulus response curve" (SRC) is used. It testsstimulus intensity-dependent recruitment of corticospinal * Correspondence: projections by means of spTMS [. The steepness of the †Equal contributors1 linear regression line through the data points of the SRC Department of Neurology, BG-Universitaetsklinikum Bergmannsheil Bochum, Buerkle-de-la-Camp-Platz 1, 44789 Bochum, Germany is a measure of corticospinal excitability [Special Full list of author information is available at the end of the article 2014 Sczesny-Kaiser et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of theCreative Commons Attribution License which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons PublicDomain Dedication waiver applies to the data made available in thisarticle, unless otherwise stated.
Sczesny-Kaiser et al. BMC Neuroscience 2014, 15:46 paired-pulse TMS (ppTMS) protocols can determine on cortical excitability and motor learning. Moreover, it intracortical facilitation (ICF) and short-latency intracorti- has not been investigated if a subsequent high frequency cal inhibition (SICI) . The normalized ICF and SICI rTMS can further increase excitability and motor learning.
ratios give information about the activity of excitatory and Here, we used a sequential 4-finger tapping/10 Hz rTMS inhibitory intracortical interneuronal circuits [].
combination paradigm previously introduced by Kim and Despite clear effects of rTMS on cortical excitability, coworkers [] combined with ATX or placebo intake in identifying consistent effects of rTMS on sensorimotor order to evaluate the effect of atomoxetine and rTMS on learning has proven more difficult. Many experiments motor cortex plasticity and motor learning in humans.
have found no changes in motor learning after high fre- Previous studies generally used neuropharmacological quency rTMS in healthy humans, while others showed only modulation only. Here, we were particularly interested mild effects Short lasting improvements, however, in possible interactional effects of both treatment regimens.
could be elicited using a combination of finger tapping task We therefore administered placebo or ATX and real rTMS or sham rTMS in a randomized double-blind study. We hy- Under these difficult circumstances and to get insight pothesized that the combination of ATX and 10 Hz rTMS of the physiology of learning processes, numerous studies over M1 is capable of increasing excitability and motor have used pharmacological interventions There are learning, and that they might have synergistic effects.
several studies on the effect of the positive allostericmodulators of the GABAA receptors, i. e. benzodiazepines ]. For example, Di Lazzaro and coworkers investi- gated the influence of lorazepam on the excitability on There were no significant differences in sex (p = 0.26) human motor cortex. They could demonstrate by means or age (mean ATX + real rTMS group: 24.1 ± 3.95 years, of single and paired-pulse TMS that lorazepam depress placebo + real rTMS group: 24.6 ± 1.74 years, ATX + sham high-amplitude motor-evoked potentials (MEP) and in- rTMS group: 24.2 ± 1.98 years, placebo + sham rTMS group: creases the excitability of inhibitory circuits Moreover, 23.9 ± 2.47 years; F(3,32) = 0.1, p = 0.96). Blood samples were it could be demonstrated that this interference with the taken from all participants approximately 1 hour and 2 hours GABAA-system can reduce learning and use-dependent after ATX or placebo (PLC) intake. The average ATX plastic changes []. Similar changes in excitability could blood serum levels in both ATX-groups were 366.1 ng/ml be demonstrated by Schwenkreis and coworkers with the after 1 hour and 296.7 ng/ml after 2 hours after drug intake.
glutamate antagonist riluzole and the NMDA antagonist Four subjects reported about temporary headache begin- memantine [. Both agents reduce intracortical facili- ning 12 hours after the experiment and persisted about 2 tation and increase intracortical inhibition. Similar to the to 4 hours. We could not distinguish if these symptoms observations of Butefisch for lorazepam, it was demon- derived from ATX or rTMS-intervention.
strated that riluzole and memantine were capable to blockuse-dependent plasticity in motor cortex.
Looking for pharmacological interventions that lead to Considering the slopes of the SRCs, repeated measurement facilitative effects and might boost learning processes, analysis of variance (rmANOVA) showed a significant the influence of norepinephrine (NE) agonists like am- effect of the within-subject factor "time" (F(1,2) = 19.26, phetamine (AMP), methyphenidate (MPH), reboxetine p < 0.000). There was no effect of the between-subject (RBX) and atomoxetine (ATX) were investigated. MPH, factor "group" (F(3,32) = 1.99, p = 0.14) or the interaction RBX and ATX increase ICF and decrease SICI measured by "time x group" (F(3,6) = 0.95, p = 0.47). Post-hoc paired paired-pulse TMS after a single dosage Moreover, t-tests revealed significantly increased slopes from T1 Plewnia and Tegenthoff investigated the modulation of (baseline measurements) to T2 (measurements 1 hour use-dependent plasticity in primary motor cortex (M1).
after ATX/placebo intake), from T2 to T3 (measurements RBX and AMP were able to enhance training-induced after motor task/rTMS combination) and from T1 to motor cortex plasticity ]. All these studies clearly show T3 (mean slope differenceΔT2T1 = 0.006 ± 0.01, t(35) = 2.96, that NE plays a crucial role in promoting plasticity. Espe- p = 0.005; mean slope differenceΔT3T2 = 0.01 ± 0.016, cially, ATX affects the regulation of NE as a highly selective t(35) = 3.7, p = 0.001; mean slope difference ΔT3T1 = 0.016 ± inhibitor of the presynaptic NE transporter with low affinity 0.02, t(35) = 5.4, p < 0.001).
for other transmitters whereas AMP and MPH act ATX-induced excitability changes between T1 and T2, as indirect NE and dopamine agonists.
being before rTMS intervention, were further assessed Considering these facts, a combination of rTMS and by pooling the results of the 2 ATX-groups and the 2 ATX would be a promising intervention that might lead placebo (PLC)-groups. Looking at the slope, rmANOVA to clear learning effects. So far, there are no placebo- with within-subject factor "time" and between-subject controlled studies on the influence of ATX and rTMS factor "group" revealed a significant effect of the factor

Sczesny-Kaiser et al. BMC Neuroscience 2014, 15:46 "time" (F(1,34) = 10.66, p < 0.00), of the interaction between No effect of either ATX or rTMS could be shown for "time" and "group" (F(1,34) = 8.57, p = 0.01) and of the factor RMT (within-subject factor "time": F(1,2) = 1.22, p = 0.3; "group" (F(1,34) = 5.09, p = 0.03), indicating a greater increase between-subject factor "group": F(3,32) = 1.78, p = 0.17; in slope in the ATX-groups as compared to the PLC- interaction "time x group": F(3,6) = 0.66, p = 0.68).
groups. For a better overview, Figure shows the meanMEP-differences of ATX- and PLC-groups between T2 and T1 (= ΔT2T1) for all 6 stimulation intensities.
Analyzing target-score (TS) data, rmANOVA demonstrated Analyzing the SICI-ratio data, rmANOVA revealed a significant effect of the within-subject factor "time" and a significant effect of the within-subject factor "time" the between-subject factor "group" (F(1,7) = 21.02, p < 0.001; (F(1,2) = 6.69, p < 0.00), no significant effect of the F(3,32) = 3.79, p = 0.02) but no significant interaction "time x between-subject factor "group" (F(3,32) = 1.14, p = 0.35) group" (F(3,21) = 0.93, p = 0.56; see Figure . Post-hoc inde- and no interaction between "time x group" (F(3,6) = 0.49, pendent two-sided t-tests revealed a significant difference p = 0.81). Post-hoc t-test showed a significant difference between PLC + sham-rTMS and ATX + real-rTMS group between T1 and T3 (meanSICI-ratio differenceΔT3T1 = (t(16) = 3.1, p = 0.007), but not for PLC + sham-rTMS 0.138 ± 0.238, t(35) = 3.5, p = 0.001). The other comparisons and ATX + sham-rTMS (t(16) = 0.6, p = 0.563) and PLC + were not significant (T1 vs. T2: t(35) = 2.1, p = 0.047; T2 vs.
sham-rTMS and PLC + real-rTMS (t(16) = 0.3, p = 0.754).
T3: t(35) = 1.9, p = 0.061).
Similar results could be shown for the execution-time RmANOVA of the ICF-ratio data showed a significant (ET)- and TSET ratio (= ratio of target score and execution effect of the within-subject factor "time" (F(1,2) = 13.9, time)-analyses (see Figure Considering ET, rmANOVA p < 0.001). Furthermore, a significant interaction be- showed significant effects for the within-subject factor tween the factor "time" and the between-subject factor "time" (F(1,7) = 47.33, p < 0.001) and the between-subject "group" could be demonstrated (F(3,6) = 2.57, p = 0.03).
factor "group" (F(3,32) = 3.3, p = 0.03) without a significant The between-subject factor "group" revealed no significant interaction between both factors (F(3,21) = 0.65, p = 0.87).
effect (F(3,32) = 2.35, p = 0.09). Post-hoc t-test showed a sig- Post-hoc independent two-sided t-tests demonstrated nificant increase of the ICF-ratio 1 hour after drug intake significant differences only between the PLC + sham-rTMS in both ATX-groups (ΔT2T1). ICF-ratio increased further and ATX + real-rTMS group (t(16) = -3.9, p = 0.001).
in the ATX + real-rTMS-group after motor training RmANOVA of the TSET ratio demonstrated a significant in- and 10 Hz rTMS (ΔT3T2). This could not be demon- fluence of the between-subject factor "group" (F(3,32) = 5.13, strated for the ATX + sham-rTMS-group. Mean ICF-ratio p = 0.01) and the within-subject factor "time" (F(1,7) = 39.14, differencesΔT2T1, ΔT3T1 and ΔT3T2 and corresponding p < 0.001). There was no significant interaction "time x p-values are shown in Table and Figure group" (F(3,21) = 1.53, p = 0.07; see Figure . Again, post Figure 1 ATX effects after 1 hour. Mean-MEP differences of the stimulus-response curves between the measurements T2 and T1. Pooled dataof ATX and PLC groups are compared. Error bars depict standard error of the mean.

Sczesny-Kaiser et al. BMC Neuroscience 2014, 15:46 Table 1 ICF-ratio data difference ± SEM difference ± SEM difference ± SEM Multiple paired two-sided post-hoc t-test of ICF-ratio data. Bonferroni-corrected p-value threshold (p = 0.025). *significant.
hoc t-test showed significant higher TSET ratio for the (see Figure Third, only the combination of ATX and ATX + real-rTMS group compared to the PLC + sham- 10 Hz rTMS significantly improved motor learning with rTMS group (t(16) = 4.4, p < 0.001). There were no sig- regard to target score and execution time (see Figure ).
nificant differences between PLC + sham-rTMS groupand PLC + real-rTMS or ATX + sham-rTMS group. No ATX led to a significant increase of corticospinal and significant differences could be shown for the ER intracortical excitability in M1 (rmANOVA: within-subject factor "time": F(1,7) = 0.01, We could reproduce the facilitative effects of ATX on p = 0.24; between-subject factor "group": F(3,32) = 1.11, cortical excitability in M1 as had been previously shown by p = 0.36; interaction "time x group": F(3,21) = 0.71, p = 0.82).
Gilbert and coworkers . In contrast, we did not see asignificant ATX-induced M1 disinhibition. The reduction of intracortical inhibition between the beginning and end of Our study yielded three major findings. First, ATX led to the study (ΔT3T1) did not depend on the group, i.e. on the a significant increase of corticospinal and intracortical type of intervention. Similar effects of NE on M1 excit- excitability in M1 one hour after intake of 60 mg ATX ability could be demonstrated for the NE reuptake in- (see Figures and ). Second, high frequency 10 Hz rTMS hibitor reboxetine. Plewnia and coworkers applied over M1 during a finger tapping motor task showed enhanced corticospinal and intracortical excitabil- was capable of further increasing intracortical excitability ity and improved motor skills in healthy subjects suggest- significantly, but only in combination with 60 mg ATX ing that this is an effect of NE reuptake inhibitors. This Figure 2 Intracortical facilitation. Treatment with 60 mg ATX significantly increased ICF after 1 hour (ΔT2T1). Further increase in ICF after rTMS/motor task sequence (ΔT3T1). p-values are shown in Table (p – threshold = 0.025). Error bars depict standard error of the mean. The absolutemean ICF-ratio values T2 from T1, T3 from T1 and T3 from T2 were subtracted (mean ICF-ratio difference ΔT2T1, mean ICF-ratio difference ΔT3T1,mean ICF-ratio difference ΔT3T1) to calculate the absolute difference. * = significant differences.

Sczesny-Kaiser et al. BMC Neuroscience 2014, 15:46 Figure 3 Motor task data. Time course of target score (TS), execution time (ET) and TS/ET-ratio (TSET) is shown for each group. The motor taskconsisted of 8 blocks. Error bars indicate standard error of the mean.
assumption could not be verified by Foster and coworkers excitability induced by ATX favors the induction of synaptic []. They found no improvement of motor learning after depression by the subsequent 10 Hz rTMS-stimulation and intake of the NE reuptake inhibitor venlafaxine compared motor task that themselves would induce LTP-like plas- to ATX [They concluded that the affinity to other ticity. Cortical LTP and LTD are typically mediated by transmitters like serotonin and the lower dosage and NMDA-receptor activation One reason, why we could the higher rate of adverse effects of venlafaxine might not see homeoplastic effects is that our 1st intervention have led to contradictory results.
(ATX intake) had no effect on NMDA-receptors but onNE-receptors. All classic homeostatic plasticity protocols High frequency 10 Hz rTMS applied over M1 during a combine rTMS, transcranial direct current stimulation finger tapping motor task was capable of further (tDCS) or paired-associative stimulation protocols that increasing intracortical excitability significantly, but only typically involves NMDA-receptors [, for example in combination with ATX 1 Hz rTMS with cathodal tDCS In our study, we wanted to extend the approach of neuro- Instead of homeostatic effects, a synergistic effect of ATX pharmacological modulation of cortical activity and its use- and rTMS was observed. The sum of gain in excitability in dependent plasticity by additionally applying 10 Hz rTMS.
the ATX + 10 Hz rTMS group could not be explained by Our rTMS paradigm itself had no significant facilitative the single effects of ATX and 10 Hz rTMS. Because higher effects on excitability parameters. This might be due to the cortical excitability is a precondition for neuronal plasticity low number of total TMS-pulses (i. e. 160 pulses). It is well and improved learning process, this finding might be closely known that rTMS effects depend on the number of total related to the fact that we observed improved motor learn- pulses, frequency and stimulation intensity [Interest- ing only after combining both interventions. The significant ingly, we could see a further increase in excitability only in increase in ICF-ratio in the PLC + sham-rTMS condition combination with 60 mg ATX (ATX + real-rTMS group).
could be explained by the motor task itself This suggests that a premedication with ATX is capableof facilitating the effects of a low pulse number rTMS Only the combination of ATX and 10 Hz rTMS improved protocol. Homeostatic plasticity did not play a role in this motor learning and execution time significantly study. Following the concept of homeostatic plasticity, we Looking at the motor task data, significant higher TS would have expected that the enhancement of motor cortex and TSET ratio and shorter ET could only be seen in Sczesny-Kaiser et al. BMC Neuroscience 2014, 15:46 the verum-verum-condition, i. e. ATX + real-rTMS group.
leads to significant increase of activity in error signaling There was no significant interaction between time and forebrain areas like bilateral inferior frontal cortex and group. Graphically, the TS, ET and TSET curves are shifted pre-supplementary motor cortex, increasing neural sen- in a parallel fashion. This indicates that the superiority sitivity for errors in healthy humans [ of the ATX + real-rTMS group was not based upon a Therefore, the combination of more effective synaptic different effect on motor learning itself but upon a better transmission within the motor system along with higher performance at the beginning of the motor task due to cognitive/behavioral sensitivity may have led to the higher cortical excitability as previously mentioned.
synergistic effect of 10 Hz rTMS and ATX seen in our Another reason might be the well known ATX effects study. It could also explain the failure of ATX or 10 Hz in promoting wakening and arousal ]. In contrast to rTMS alone to be effective.
the results of Kim and coworkers , we did not observehigher TS for the PLC + real-rTMS group compared to Pharmacological interventions and rTMS the PLC + sham-rTMS. This may be due to our modified Studies combining rTMS and neuropharmacological and easier motor task, which could have prevented smaller intervention were usually undertaken to investigate the differences in learning to become visible.
role of transmitters in the induction of rTMS after-effects Considering detail of our motor task, we decided to and not to boost performance like we did in our study.
choose the non-dominant hand and according non- Huang et al. combined a specialized rTMS-protocol called dominant hemisphere (left hand, right hemisphere) theta-burst-stimulation (TBS) with the NMDA receptor for motor task, excitability measurements and rTMS- antagonist memantine They found that, on one hand, intervention because we wanted to avoid a ceiling effect memantine inhibited the facilitatory effect of intermittent We did not test the excitability parameters of the TBS (iTBS) on MEP amplitudes. On the other hand, it contralateral hemisphere and motor learning of the blocked the suppressive effect of continuous (cTBS). Teo contralateral hand. Thus, previous studies could dem- et al. used the NMDA receptor coagonist D-cycloserine onstrate that there is a hemispheric asymmetry of cor- that acts at the glycine site of the NMDA receptor. They ticospinal activation with a higher MEP facilitation for found that it reversed the aftereffect of iTBS from facilita- the non-dominant (left) hemisphere [Brouwer tion to inhibition Lang et al. performed a 1 Hz rTMS et al. showed that this different level of excitation is study using the dopamine receptor agonist pergolide and not related to speed or dexterity of finger movements.
found that the suppression of corticospinal excitability by Relating to our study, we would expect similar facilitative rTMS was more pronounced after drug intake compared effects of ATX and rTMS on dominant hemisphere.
to placebo [These results show in general that NMDA We also can purpose that there were differences in and dopaminergic receptors play a role in the induction of levels of cortical excitability between hemispheres not rTMS effects.
only in baseline but also later in postinterventional So far, no study has been undertaken to investigate measurements. But according to Brouwer's results, these adrenergic influences on rTMS effects. Furthermore, we differences had no relation to differences in motor per- have not found any study that considers the combination of formance between both hands.
pharmacological intervention, rTMS and motor learning.
Interestingly, motor learning was not affected by ATX In this case, we describe synergistic effects between rTMS or 10 Hz rTMS alone. According to the subtraction and pharmacological modulation for the first time.
method devised by Donders the entire effects on TSor ET should be explained by the cooperativity of the ATX and rTMS effects. Applying this method, a factor "X" Considering some limitations of our study, we surely have remains which cannot be explained by the separate ATX to mention the relative low number of subject per group and rTMS effects (see Figure . This kind of synergistic (n = 9). Furthermore, we did not choose a cross-over effect "ATX x rTMS" indicates nonlinear interaction design, which would have allowed intra-subjects compari- between both components. The neuronal basis of this son. A cross-over design would have had advantageous for interaction, however, remains unclear. High frequency the interpretation of the results, rendering out biases that rTMS activates the motor network including premotor comes from inter-individual variability of cortical excit- cortex, supplemental motor area and ipsilateral primary ability, partly determined by brain morphology .
motor cortex, and increases effective connectivity between Moreover, one might argue that the use of circular coil these areas [The finger tapping task itself activates for excitability measurement does not extract reliable mea- a similar system including contralateral primary sensori- sures compared to a figure-of-eight coil. Ugawa et al. could motor cortex, premotor cortex, supplementary motor demonstrate no different results between figure-of-eight cortex, bilateral secondary somatosensory areas and basal coil over left hand motor area and circular coil over ver- ganglia and ipsilateral cerebellum In addition, ATX tex for the determination of corticocortical facilitation Sczesny-Kaiser et al. BMC Neuroscience 2014, 15:46 Figure 4 Factorial design. This graphic shows the idea of a synergistic effect that only occurs in the verum-verum-group D (combination ofboth interventions). Furthermore, it shows how to calculate the synergistic effect. Group A = PLC + sham-rTMS; group B = PLC + real-rTMS; groupC = ATX + sham-rTMS; group D = ATX + real-rTMS. X indicates interaction factor "ATX x real-rTMS" (synergistic effect).
and inhibition Moreover, in our TMS-studies, we pre- (DRKS-ID: DRKS00004653). All subjects were right-handed fer the use of the circular coil. We could see stable results as revealed by the Edinburgh Handedness Inventory ].
and the positioning of this coil is less critical They all denied the practice of fine motor skills presently orin the past, such as playing a guitar or piano or as having experience in professional typewriting. All participants were Previous studies could show that high frequency rTMS free of medication.
and ATX are capable to modulate cortical plasticity andto improve motor learning Possible interaction effects Time course and study design have never been investigated. In the present study, we could The study was randomized and placebo-controlled. It show that the combination of a pharmacologically-induced was double-blind for the ATX vs. PLC condition and increase in NE transmission and 10 Hz rTMS exerts syner- single-blind for the rTMS and sham-rTMS condition.
gistic effects on cortical excitability and motor learning in A single dose of 60 mg ATX or placebo was given after healthy humans.
baseline excitability measurements (T1). Blood samples This could be a promising approach to improve motor were collected one and two hours after administration learning in patients with neurological disorders like of ATX or PLC to determine ATX plasma concentration.
stroke, traumatic brain injury and neuromuscular diseases One hour after drug intake, excitability measurements were (e. g. amyotrophic lateral sclerosis). Especially, it would repeated (T2). Then, all participants performed a sequential be interesting to investigate the development and consoli- motor task with their non-dominant left hand while inter- dation of neuronal plasticity effects in primary motor cor- mittently receiving single-blinded real 10 Hz rTMS or tex when rTMS and ATX were administered over several sham-rTMS in the manner described below. Finally, excit- days. Furthermore, it remains unclear if such effects would ability parameters were measured again (T3). Each meas- be seen with other facilitative drugs like modafinil or urement session took approximately 30 to 40 minutes, the amantadine or in combination with other brain stimu- rTMS + motor practice 10 minutes, and the entire study lation protocols like tDCS and TBS.
three hours (see Figure Participants left TMS-laboratory after baseline measure- Data from 36 healthy subjects (19 women, 17 men) were ments and went to a special room where the non-blinded collected and analyzed. Subjects were randomly assigned examiner (P.S.) who was participated in randomization and to four equally-sized groups (n = 9). All subjects gave their administration of the drugs only delivered the capsules.
written informed consent. The protocol was approved Subjects received either a yellow-blue 60 mg ATX capsule by the local ethical committee of the Ruhr-University (Eli Lilly™) or a yellow-red capsule with mannitol (placebo).
of Bochum (registry no. 4317-12) and was performed They swallowed it with a drink of water in this room and in accordance with the Declaration of Helsinki. The stayed for one hour before returning in TMS-laboratory.
study is registered in German Clinical Trials Register The TMS-examiners have seen neither the capsules nor the

Sczesny-Kaiser et al. BMC Neuroscience 2014, 15:46 Figure 5 Time course of the study and study design.
drug intake itself. The subjects and TMS-examiner did not Stimulus-response curve know color code.
For SRC, spTMS was applied at 100%, 110%, 120%, 130%,140% and 150% of individual RMT. For each stimulus Excitability measurements by TMS intensity, 12 trials were performed. We used the same The following parameters of corticospinal and intracortical TMS-setting for SRC as for RMT-determination. For excitability in the primary motor cortex were investigated: analysis of the SRC data, the slope between data points resting motor threshold (RMT), stimulus-response curve Ti, i =1, 2, 3 of the SRC as the steepness of the linear re- and both short intracortical inhibition and intracortical gression line through the given data points (between facilitation, assessed using paired-pulse TMS [MEPs 100% and 150% stimulation intensity of the individual were recorded with Ag-AgCl-surface electrodes using a RMT) was calculated ].
standard electromyography device (Neuropack M1; NihonKohden, Tokyo, Japan). While stimulating the contralateral hemisphere, recordings were taken from the left first dorsal To apply ppTMS, the circular coil was connected to the interosseus muscle (FDI). The signals were recorded with a Bistim® device which triggered two Magstim 200® stimu- sampling rate of 5 kHz, and amplified with a bandpass of lators. Earlier studies had shown that focal and circular 20 Hz - 3 kHz, a sweep duration of 10 - 50 ms/div and coils elicited comparable results in pp-TMS studies a gain of 0.1 mV/div. During the entire measurements, In this ppTMS assembly, a separate RMT had to be de- muscle relaxation was monitored by EMG. Subjects were termined because of the lowered stimulator output in seated in a comfortable chair in a silent and bright room.
case the Magstim 200 is connected to the Bistim® device.
We tested the interstimulus intervals (ISI) 2, 4, 10 and Resting motor threshold 15 ms. The second stimulus (test stimulus) was adjusted RMT was determined with single-pulse TMS to the to evoke an MEP of approximately 1.0 mV. The condi- nearest 1% of the stimulator output, and was defined tioning stimulus was set at 80% of the individual ppTMS as the minimum intensity which produced four motor assembly RMT. For each ISI, 12 trials were performed.
evoked potentials > 50 μV out of eight trials Single- Before and after ppTMS, 12 single control stimuli using pulse TMS was applied using a Magstim 200® stimulator the same stimulation intensity as for the second (test) (Magstim, Whitland, Dyfed, U.K.) connected to a circular stimulus were applied. The amplitude ratio of the mean coil (outer diameter 14 cm). The coil was placed with its conditioned MEP to the mean control MEP was calculated center near the vertex with the current flowing clockwise for each ISI. For further statistical analysis, parameters in the coil in order to activate predominantly the right of SICI and ICF were defined as the averages of MEP hemisphere and to produce MEP in the left FDI. This ratios obtained at inhibitory ISIs of 2 and 4 ms (SICI), position was marked with a red wax pencil to improve and at facilitative ISIs of 10 and 15 ms (ICF) reproducibility of placement.
ICF-ratios between the three time points were compared

Sczesny-Kaiser et al. BMC Neuroscience 2014, 15:46 by subtracting the means (mean ICF-ratio differenceΔT2T1, spTMS elicited the highest MEP amplitude (hotspot of mean ICF-ratio differenceΔT3T1 and mean ICF-ratio the FDI). At the FDI hotspot, we had to determine a new RMT because we used a figure-of-eight coil for focalrTMS. Twenty pulses of 10 Hz rTMS were applied for 2 seconds just prior to the beginning of each task block A modified combined session of motor task and 10 Hz with an intensity of 80% of individual RMT. A total of rTMS as previously described by Kim and coworkers 160 pulses were given during each experiment consisting of was used. The task was designed using Presentation® eight task blocks. For sham-rTMS, the same stimulation software (Neurobehavioral Systems Inc., Albany, California, parameters and the same figure-of-eight-coil were used, USA). Subjects were seated in a comfortable chair and except for the stimulation intensity, which was set at the placed 35 cm in front of a 19 inch LED-monitor. A seven- lowest possible stimulation intensity (10% of maximal digit sequence of numbers, namely the combination of "1 2 stimulator output). Previous work of ours confirmed 3 4 1 2 3" was presented on the monitor for 40 seconds.
this intensity to have only local effects at the scalp with no Subjects were instructed to repeatedly push the four num- effects on neuronal excitability in the motor cortex ].
bered buttons of the response box (Lumitouch™, PhotonControl, Burnaby, Canada) as accurately, as quickly and as ATX serum concentration often as possible using their left hand (one task block).
Blood samples were taken from all participants approxi- In consideration of this quite easy tapping task, we de- mately 1 hour and 2 hours after drug or PLC intake. After cided to choose the non-dominant and non-specialized finishing the study, ATX serum concentration was deter- left hand to ensure motor improvement and learning mined by liquid-chromatography tandem mass spectrom- and to avoid an early ceiling effect. Our decision was etry method (Laboratoriumsmedizin Dr. Eberhard & Co., not based on electrophysiological aspects like asymmetry Dortmund, Germany).
in corticospinal activation but on motor task aspects.
Each button relates to one finger (no. 1 to the index finger,no. 2 to the middle finger, no. 3 to the ring finger, and no.
Statistical analysis 4 to the little finger). The motor task required the repetition RmANOVA was performed with the within-subject factor of eight identical task blocks with pauses of 28 seconds "time" and between-subject factor "group" to assess interleaved (see Figure Within each task block, motor differences between different points of measurement practice was preceded by rTMS application. Before starting for the excitability parameters. Univariate ANOVA was the entire motor task, subjects were allowed to perform performed using age as dependent variables and group two blocks of practice to familiarize themselves with the as the between-subject factor. A chi-squared test was equipment and experimental procedure. In this case, we used to analyze sex differences between the different used a different combination of numbers.
groups. For analysis of the behavioral data, we consideredthe total number of correct responses of an entire 7-digit sequence (target score = TS), the execution time (ET) For rTMS application, a Magstim Rapid® stimulator defined by the time required to the subsequent correct (Magstim, Whitland, Dyfed, UK) and a figure-of-eight response, the ratio of TS and ET (TSET) and the error shaped coil (outside diameter 8.7 cm, peak magnetic rate (ER) defined by the equation field strength 2.2 T, peak electric field strength 660 V/m)were used. The coil predominantly stimulates neuralstructures below the junction of the two coils. During the entire stimulation procedure the coil was held tangen- tially to the head in posterior-anterior direction with thehandle pointing backwards.
Real- and sham-rTMS were delivered over the right RmANOVA was performed with within-subject factor motor cortex at the scalp position where suprathreshold "time" and between-subject factor "group". Where it was Figure 6 Design of rTMS/motor-task sequence.
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doi:10.1186/1471-2202-15-46Cite this article as: Sczesny-Kaiser et al.: Synergistic effects of noradrenergicmodulation with atomoxetine and 10 Hz repetitive transcranial magneticstimulation on motor learning in healthy humans. BMC Neuroscience 2014 15:46.
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