Short- and intermediate-interval cortical inhibition and facilitation assessed by navigated transcranial magnetic stimulation

Journal of Neuroscience Methods 195 (2011) 241–248

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Short- and intermediate-interval cortical inhibition and facilitation assessed by navigated transcranial magnetic stimulation夽,夽夽 Laura Säisänen a,b,∗ , Petro Julkunen a,c , Eini Niskanen a,d , Taina Hukkanen a , Esa Mervaala a,b , Jari Karhu e,f , Mervi Könönen a,c a

Department of Clinical Neurophysiology, Kuopio University Hospital, Kuopio, Finland Department of Clinical Neurophysiology, Institute of Clinical Medicine, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland Department of Clinical Radiology, Kuopio University Hospital, Kuopio, Finland d Department of Applied Physics, Faculty of Science and Forestry, University of Eastern Finland, Kuopio, Kuopio, Finland e Nexstim Ltd, Helsinki, Finland f Department of Physiology, Institute of Clinical Medicine, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland b c

a r t i c l e

i n f o

Article history: Received 12 April 2010 Received in revised form 19 November 2010 Accepted 28 November 2010 Keywords: Navigated transcranial magnetic stimulation Paired pulses Short-interval intracortical inhibition Intracortical facilitation Motor evoked potential

a b s t r a c t Objective: To characterize the behaviour of primary motor cortex and to determine appropriate measurement parameters for short-interval cortical inhibition (SICI) and intracortical facilitation (ICF) by paired-pulse transcranial magnetic stimulation (TMS) with the aid of MRI-based neuronavigation. Methods: Paired-pulse TMS was targeted to the optimal cortical representation sites of the abductor pollicis brevis (APB) muscle in 48 healthy right-handed volunteers. Motor evoked potentials (MEPs) were recorded from the APB and abductor digiti minimi (ADM) muscles. The conditioning stimulus (CS) intensities were 80% and 90% and the test stimulus was 120% of the resting motor threshold (rMT). The interstimulus intervals (ISIs) were 3, 7, 13, 22 and 28 ms. Results: Inhibition was observed at 3 ms with a CS of 80%. Facilitation emerged at ISIs of 7 and 13 ms with both CS intensities, more prominently with 90%. At ISI of 22 ms, facilitation was observed in ADM (p < 0.01) but not in APB. No uniform amplitude change was observed at ISI of 28 ms. For both muscles, MEP latencies were shortened (p < 0.01) at ISIs of 3 and 7 ms and prolonged (p < 0.01) at 28 ms. Conclusions: Inhibition is most prominent at ISI of 3 ms and CS of 80% of rMT, whereas CS of 90% of rMT and ISIs of 7 and 13 ms are preferable for facilitation. Latencies appear to be stable and independent indicators of both phenomena should be taken into account. Significance: Both the latency and amplitude of MEPs are important parameters when paired-pulse paradigms are used in clinical studies. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Motor cortex excitability can be studied by transcranial magnetic stimulation (TMS) utilizing the paired-pulse (PP) technique. In the conventional PP method, two consecutive magnetic stimuli, a subthreshold conditioning stimulus (CS) and a following test stimulus (TS) with an intensity above the individual resting motor threshold (rMT) are delivered through a TMS coil over motor cortical region (Claus et al., 1992; Kujirai et al., 1993; Ziemann, 1999). The behaviour of conditioned motor evoked potentials (PP-MEPs)

夽 Work was done in Kuopio University Hospital, Kuopio, Finland. 夽夽 Financial interests: Jari Karhu works as part-time chief medical officer of Nexstim Ltd. ∗ Corresponding author at: Department of Clinical Neurophysiology, Kuopio University Hospital, P.O. Box 1777, FI-70211 Kuopio, Finland. Tel.: +358 17 174115; fax: +358 17 173244. E-mail address: laura.saisanen@kuh.fi (L. Säisänen). 0165-0270/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2010.11.022

is believed to reflect the function of inhibitory and excitatory interneurons by activating individual cortical circuits within the stimulated motor cortex. The modulatory effect of the CS on the amplitude of the TS evoked MEPs depends mostly on two factors: the intensities of the CS and TS, and the interstimulus interval (ISI) between the two stimuli. Short interval intracortical inhibition (SICI) occurs at ISIs of 1–5 ms, and intracortical facilitation (ICF) at ISIs of 7–20 ms (Kujirai et al., 1993). However, recent studies have revealed that SICI consists of at least two phases of inhibition which are physiologically distinct (Hanajima and Ugawa, 2008). As well, SICI and ICF are to some extent overlapping, depending on ISIs and stimulus intensities (Reis et al., 2008). In the PP paradigms usually the PP-MEP amplitude is studied, but changes in latency, have recently been reported as well (Kossev et al., 2003; Shimizu et al., 1999). Alterations of this well-known SICI–ICF pattern have been observed in various neurological conditions (Hanajima and Ugawa, 2008). Cortical plasticity can be studied with the PP method

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(Bütefisch et al., 2006; Cohen et al., 1998; Rosenkranz et al., 2007). In particular, reduced SICI has been observed in patients with stroke (Berweck et al., 2008). Furthermore, the PP method is suitable for the study of the effects of various neurotransmission modulating drugs (Kapogiannis and Wassermann, 2008; Ziemann, 2004). Unfortunately, the results have not been consistent and the notoriously high normal variability of MEPs has prevented widespread use of PP in clinical context. As supported by our earlier study conducted with single-pulse TMS, MRI-based navigated TMS (nTMS) should be an optimal tool for inducing reproducible PP-responses as nTMS delivers stimuli with higher spatial accuracy and produces responses with greater reproducibility than the conventional, non-navigated TMS, resulting in lower intra-individual variation in responses (Julkunen et al., 2009). Here we performed an observational study with no a priori hypotheses concerning the effect of navigation. The aim of the present study was to characterize PP-MEP latencies and amplitudes for both SICI and ICF in an exploratory manner when stimulus delivery was controlled by nTMS. nTMS allowed on-line navigation relative to individual anatomical structures, targeting the stimuli, and repeating the magnetic pulses to a certain location in the motor cortex accurately (Ruohonen and Karhu, 2010). We used two intensities for CS and several ISIs in a large population of healthy subjects in different age groups. The ISIs were those commonly used for induction of SICI and ICF based on earlier non-navigated studies so that the data would be fully comparable with those gained by conventional TMS. We chose ISI of 3 ms for inducing SICI, ISIs of 7 ms and 13 ms for inducing ICF and additionally ISIs of 22 ms and 28 ms to observe decline in ICF. ISIs of both 7 and 13 ms were used for ICF to detect any difference between them. The possible effects of age, gender and hemisphere were also investigated. Our overall goal was to determine appropriate measurement parameters for inducing SICI and ICF for their assessment in clinical studies. 2. Methods We studied 48 healthy right-handed volunteers (26 females and 22 males, mean age 49 years, age range 22–79 years, eight subjects per decade) with nTMS. None of the subjects had any central nervous system (CNS) diseases or psychiatric disorders and none were on medications with known CNS effects, or had any commonly accepted contraindications to TMS. The study was performed according to the standards of the latest revision of the Declaration of Helsinki. All subjects gave their written informed consent prior to participating and the study was approved by the local ethical committee of Kuopio University Hospital, Kuopio, Finland. Handedness of the subjects was determined according to the Waterloo Handedness Questionnaire – revised and reduced form with 20 items (Elias et al., 1998) as well as subjects’ own opinion (for details, see Niskanen et al., 2010; Säisänen et al., 2008). 2.1. Measurement setup Three-dimensional head models were constructed in the navigation software (eXimia 2.2, Nexstim Ltd, Helsinki Finland) using individual MR-images. The stimulation setup consisted of the tracking unit (Passive Polaris Spectra, NDI) combined with a magnetic stimulator (Nexstim Ltd, Helsinki, Finland) and focal figure-of-eight TMS coil with monophasic pulse type (mean wing radius of 59 mm). During stimulation, electromyography (EMG) was recorded and monitored continuously on-line (ME 6000, Mega Electronics Ltd, Kuopio, Finland) using pre-gelled disposable Ag/AgCl electrodes. Active electrodes were attached to the skin overlying the abductor pollicis brevis (APB) and abductor digiti minimi (ADM) muscles.

Reference electrodes were placed on the metacarpophalangeal joint. The EMG signals were band-pass filtered (8–500 Hz), amplified, displayed and stored for off-line analysis. 2.2. Determination of optimal site and motor threshold The cortical representation of the hand muscles at and around the anatomically defined motor “hand knob” (Yousry et al., 1997) was extensively mapped with a stimulation intensity eliciting MEPs of around 300 ␮V, while the stimulus-induced current (E-field) on the cortex was oriented perpendicularly to the anatomically defined central sulcus. The site which evoked MEPs with highest amplitude at APB was then targeted, and the coil was rotated horizontally to find the optimal orientation of induced current, i.e., providing repeatedly the largest MEP. Using this coil location and orientation (the optimal stimulation site), the rMT was determined as the intensity inducing five responses (≥50 ␮V peak-to-peak) out of ten consecutive pulses given 5–10 s apart (Chen et al., 2008; Rossini et al., 1994). Suggestion for the MT was first achieved with aid of a 10 stimuli using an automated threshold hunting protocol (Awiszus, 2003). The suggested MT was verified by 10 stimuli, of which optimally, 5 responses were induced. If not, the stimulus intensity was changed in steps of 1% stimulator output to ultimately achieve an MT which provided responses with at least 5 times of 10. Absence of background muscle activity was ensured by continuous visual monitoring of spontaneous EMG. Ten single-pulse stimuli (non-conditioned, control MEP response) were induced at the intensity of 120% of rMT to determine the baseline at the optimal APB representation. 2.3. Paired-pulse paradigm The TMS pulses were targeted to the previously determined hotspot with <2 mm deviation in coil location using the aiming tool implemented in eXimia software. PP paradigm included a subthreshold conditioning stimulus (CS, 80% and 90% related to each subject’s individual rMT) and a suprathreshold test stimulus (TS, 120% of rMT) with five ISIs (3, 7, 13, 22 and 28 ms) with random inter-trial interval of 5–10 s. Five responses were collected for each CS intensity and ISI. The order of the hemisphere first stimulated was also randomized across subjects. In the cases where the subject’s rMT was higher than 83% of the maximal stimulator output and thus preventing to achieve 120% of rMT (n = 6), the maximum of the stimulator output was used for TS intensity. 2.4. Data analysis In order to prevent spontaneous muscle activity from influencing SICI or ICF (Garry and Thomson, 2009; Ridding et al., 1995), only MEPs without any muscle activation preceding the stimuli were included in the analysis. Negligible, involuntary muscle activity of less than ±25 ␮V at 50 ms before was deemed acceptable. The muscle responses were analyzed using the MegaWin software (Mega Electronics Ltd, Kuopio, Finland). The control MEP amplitude and latency were defined as the mean of ten trials at intensity of 120% of rMT. PP-MEP latency and amplitude analysis were performed using the individual mean value for each CS and ISI calculated from the five trials. Because of the high variability of single responses, the highest and lowest amplitude MEPs for each PP condition were excluded before averaging. Also, the normalized amplitudes are given as median of five trials for comparison. Latency was defined by inspecting each MEP trace visually and placing the marker manually to the first abrupt deflection with sufficient size and repeatable waveform from the baseline that is directly linked to the highest peak-to-peak amplitude without any delay. The amplitude was measured peak-to-peak (Fig. 1). The EMG

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Fig. 1. A representative sample of EMG curves for control MEP and all tested ISIs at CS 80% of MT. APB is shown on the left and ADM on the right column. The stimulation moment is indicated as a dashed grey line. The single pulse MEP is an averaged trace of ten pulses. For each PP-MEP three traces (lowest and highest excluded) are shown. Respective ISI is indicated in the upper right corner of each box.

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Fig. 2. Difference between conditioned and control MEP amplitudes with different ISIs. Significant difference, p < 0.01, is indicated with an asterisk. Error bars indicate 95% CI for the difference. Results are represented for both measured muscles (APB on the top, ADM below) and both stimulated hemispheres.

signal from the relaxed muscle 50 ms prior to stimulus was considered as the baseline EMG. 2.5. Statistical tests Linear mixed model with Sidak adjustment for multiple comparisons was used in the statistical analysis to test the effects of different variables. PP-MEP amplitude was set as dependent variable and hemisphere, gender, age, studied muscle and CS intensity were used as fixed factors with subject as random factor. The assumption of the model (normality of residuals) was not fulfilled for normalized MEP responses. Thus, raw data MEP amplitudes were examined with linear mixed model. In addition, normalized values for amplitude were used and were compared between different size MEPs (<750 ␮V, 750–2000 ␮V or >2.0 mV) separately with each ISI, hemisphere and CS using non-parametric Mann–Whitney U test. The rMT and control MEP amplitude differences between hemispheres were tested using independent-samples t-test. For testing how reliably SICI and ICF would appear, amplitude and latency analyses were performed using paired-samples t-test to compare each subject’s PP-MEP to the individual control MEP. Statistical analyses were performed with SPSS (version 17.0, Chicago, IL, USA).

3. Results The CS intensity, stimulated hemisphere and recorded muscle exhibited significant (p < 0.01) effects on the PP-MEP amplitudes. No significant effect was found for rMT, gender, age or stimulated hemisphere. Interaction effects were not observed. A representative set of MEPs (control and PP) is presented in Fig. 1. There were no inter-hemispheric differences in rMT or control MEP parameters (Table 1). 3.1. PP-MEP amplitude SICI and ICF were quite similar in both the optimally targeted APB and in the cortically adjacent ADM muscles. SICI was induced at ISI of 3 ms with CS of 80% but not with CS of 90% of rMT (Fig. 2). ICF was evident at ISI of 7 ms and maximal at ISI of 13 ms with both CS intensities being more pronounced at CS of 90% of rMT (Fig. 2). Pairwise comparisons with Sidak adjustment did not show significant differences between ISIs 7 and 13 ms. At ISI of 22 ms, the facilitation started to diminish and ICF was significant (p < 0.01) only in ADM. At ISI of 28 ms no uniform change in amplitude was observed. SICI was weaker and ICF stronger at small test MEPs (<750 ␮V) compared to larger ones (Fig. 3).

Table 1 Motor thresholds and control MEP characteristics. Mean ± SD. Both hemispheres were stimulated and MEPs were measured in two separate muscles.

MT (%) MEP latency (ms) MEP amplitude (␮V)

Muscle

Left hemisphere

APB APB ADM APB ADM

64 22.2 22.2 852 659

± ± ± ± ±

13 1.8 2.1 753 604

MT = motor threshold (normalized to the maximum stimulator output); MEP = motor evoked potential.

Right hemisphere 65 22.7 22.3 989 829

± ± ± ± ±

13 1.8 1.8 898 823

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µ

245

µ

Fig. 3. The effect of test MEP size on SICI and ICF. Results are represented for both hemispheres and both intensities (80% of rMT on the top and 90% of rMT below) for the APB. Error bars show 95% confidence interval. Large standard deviation in large responses (>2 mV) is due to low number of responses, which in few cases was too low for revealing statistical differences. Statistically significant differences between MEP sizes are indicated (*p < 0.05).

SICI emerged at CS of 80% of MT in either APB or ADM in 91.7% of the subjects in both hemispheres. Similarly, at CS of 90% of rMT, ICF was visible in 82.0% at ISIs 7 ms and in 84.3% at 13 ms. The subjects in whom ICF was not exhibited were not the ones with high rMT. 3.2. PP-MEP latency Significantly shortened (p < 0.01) MEP latencies were observed at ISIs of 3 and 7 ms (Fig. 4, Table 2) when compared to the control

MEP. ISI of 13 ms proved to be a turning point in the behaviour of latencies, which were significantly prolonged at 28 ms (p < 0.01). CS of 90% had significantly stronger impact on shortening latency (p < 0.05) than 80%. At ISI of 3 ms latency shortening was observed in 86.5% of the subjects at CS of 80% of rMT and in 88.6% at CS of 90% of rMT. When using CS of 90% of rMT, shortened latencies at ISI of 7 ms were observed in 92.7% of the subjects. Prolonged latencies at ISI of 28 ms were observed in 86.7% of the sub-

Table 2 Latency differences between conditioned and control MEP and normalized amplitudes in APB using mean value (highest and lowest values were excluded) and median with 95% confidence interval (lower–upper boundaries). ISI

Left hemisphere Latency difference (ms)

Right hemisphere Normalized amplitude (mean)

Conditioning stimulus intensity of 80% of resting motor threshold 3 −0.83 (−1.23 to −0.44) 0.67 (0.47–0.88) 7 −0.73 (−1.03 to −0.43) 2.00 (1.44–2.57) 13 −0.05 (−0.55 to 0.46) 1.80 (1.35–2.25) 22 0.35 (0.06–0.65) 1.20 (0.90–1.51) 28 0.69 (0.36–1.01) 0.97 (0.72–1.22) Conditioning stimulus intensity of 90% of resting motor threshold 3 −1.53 (−1.95 to −1.12) 1.09 (0.80–1.37) 7 −1.19 (−1.56 to −0.82) 1.98 (1.56–2.41) 13 −0.37 (−0.73 to −0.09) 2.17 (1.44–2.91) 22 0.32 (−0.04 to 0.68) 1.61 (0.96–2.28) 28 0.47 (0.06–0.87) 0.77 (0.50–1.03) ISI = interstimulus interval.

Normalized amplitude (median)

Latency difference (ms)

Normalized amplitude (mean)

Normalized amplitude (median)

0.56 (0.37–0.76) 1.80 (1.40–2.20) 1.70 (1.29–2.12) 1.24 (0.94–1.53) 1.07 (0.79–1.34)

−0.73 (−1.07 to −0.40) −0.61 (−0.93 to −0.29) 0.06 (−0.31 to 0.43) 0.43 (0.09–0.77) 0.68 (0.30–1.05)

0.83 (0.39–1.27) 1.76 (1.37–2.14) 2.02 (1.53–2.51) 1.49 (1.16–1.82) 1.38 (1.04–1.71)

0.61 (0.30–0.92) 1.86 (1.30–2.42) 1.89 (1.41–2.38) 1.36 (1.08–1.64) 1.32 (0.97–1.66)

1.21 (0.82–1.60) 2.02 (1.57–2.46) 2.27 (1.57–2.97) 1.50 (0.92–2.08) 0.84 (0.40–1.28)

−1.51 (−1.88 to −1.13) −1.40 (−1.81 to −0.99) −0.33 (−0.76 to 0.10) 0.39 (−0.13 to 0.91) 0.58 (0.03–1.12)

1.66 (1.02–2.29) 2.21 (1.55–2.86) 3.02 (1.81–4.23) 1.87 (0.99–2.74) 1.34 (0.81–1.88)

0.92 (0.60–1.25) 2.11 (1.53–2.69) 2.41 (1.50–3.33) 1.56 (0.95–2.16) 1.15 (0.71–1.59)

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Fig. 4. Difference between conditioned and control MEP latencies with different ISIs. Significant difference, p < 0.01, is indicated with an asterisk. Error bars indicate 95% CI for the difference. Results are represented for both measured muscles (APB on the top, ADM below) and both stimulated hemispheres.

jects using CS of 80% of rMT and in 84.6% using CS of 90% of rMT. 4. Discussion We characterized the PP-MEP responses by MRI-based navigation of TMS. According to the present results, the combined analysis of amplitude and latency for both SICI and ICF adds to the information content and may provide new views to intracortical inhibition–excitation balance using the PP method. By determining the difference between PP-MEP and control MEP latency using a 95% confidence interval, the response can be classified as normal or deviating (Fig. 4). The present results that are obtained with the possibly most accurate model presently available and with a reasonable large number of healthy subjects encourage the clinical use of this method. When the stimuli were targeted to the individually optimal cortical representation of APB, SICI was prominent at ISI of 3 ms (CS of 80% of rMT) whereas ICF was clear at ISI 7 ms and maximal at ISI 13 ms (with both CS intensities 80% and 90% of rMT). These findings corroborate those of previous studies (Boroojerdi et al., 2000b; Chen et al., 1998; Civardi et al., 2000; Kossev et al., 2003; Kujirai et al., 1993; Liepert et al., 2001; Maeda et al., 2002; Orth et al., 2003). In our study, the phenomena were observed in >90% of the subjects. The test MEP size also affected SICI and ICF, i.e., weaker SICI and stronger ICF were observed with small test MEPs in accordance to previous studies (Fig. 3) (Daskalakis et al., 2002; Ferreri et al., 2011; Wagle-Shukla et al., 2009). Age and gender did not correlate with rMT. The PP-MEP latencies, which have not been extensively studied, may be sensitive additional markers of SICI and ICF according to the

present study. We observed shortened latencies at ISIs of 3 and 7 ms and prolonged latencies at 28 ms (Fig. 4). Shortened PP-MEP latencies have been observed earlier (Kossev et al., 2003; Shimizu et al., 1999), and proposed as a more sensitive indicator for ICF than MEP area with hypothesis of a shorter pathway (involving less synapses) for ICF than for SICI (Kossev et al., 2003). As well, shortened latency for SICI has also been observed (Kujirai et al., 1993). Somewhat surprisingly, the latencies were consistently longer for both APB and ADM at ISI of 28 ms. To our knowledge, no changes in latency at ISIs longer than 20 ms have been previously described. The mechanism for the prolonged latency at 28 ms can only be speculated on; a plausible explanation would be a spinal mechanism, for example an inhibition of a lower motoneuron by the CS. Although previous studies have shown that CS does not change the spinal excitability measured by H-reflex (Kujirai et al., 1993), spinal mechanisms and inhibition of lower motoneuron pool cannot be excluded with certainty. Furthermore, for instance teeth clenching, having a facilitatory effect, has been shown to have both cortical and subcortical sites for modifying hand muscle activity (Boroojerdi et al., 2000a). Epidural recordings have shown that a single TS produces 3–4 waves of inhibitory activity (indirect, I-waves) reflecting descending spinal cord volleys whereas subthreshold CS evokes no such recognisable descending activity (Di Lazzaro et al., 1998; Nakamura et al., 1997). All these volleys contribute to the peak-to-peak size of the peripheral EMG response. As the first I1-wave is not modified by the CS, it strongly suggests that CS does not modify directly the excitability of pyramidal cells, but elicits synaptic inhibition in the motor cortical circuits (Di Lazzaro et al., 1998). This, in turn, makes it unlikely that the inhibition at ISI of 3 ms is due to refractory corticospinal axons and/or after hyperpolarization in the cell body. The inhibition is evoked especially in I3-wave at ISIs 3–5 s (Hanajima

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et al., 2003; Shirota et al., 2010). Based on these earlier investigations the observed latency change in our study at ISI of 3 ms may be associated with suppression of I3-waves. SICI is a complex phenomenon and according to recent studies it is contaminated and overlapped with ICF at certain ISIs and CSs (Peurala et al., 2008). To measure pure GABAergic inhibitory system instead of net inhibition, ISIs of 1.5–2.0 ms (Ziemann et al., 1998) or 3–5 ms (Hanajima et al., 2003; Vucic et al., 2009) are preferable. ISIs between 2.1 and 2.6 ms have been proposed as most effective (Fisher et al., 2002; Ilic et al., 2002; Orth et al., 2003; Roshan et al., 2003; Vucic et al., 2009), but these ISIs are in the range of concurrent facilitation. As CS intensity approaches the rMT, the facilitation of spinal motoneurons likely contributes (Hanajima and Ugawa, 2008) and that is why lower CS intensities (70–80% of rMT approximately equivalent to 90–100% of active motor threshold, aMT) should be used (Hanajima and Ugawa, 2008). Thus, a limitation of this study is that we only used two different CSs whereof 90% of rMT probably was too high and might have modified descending volleys to spinal interneurons and possibly to alpha-motoneurons and subsequently influenced the latencies. However, using aMT to ensure that CS is subthreshold, which is currently popular method, would have required standardizing the level of voluntary activation, and thus might have added more variation to the results. It has been studied whether SICI is dependent on the intensity of the TS or the size of the test MEP and how TS should be determined, as percentage of rMT or MEP of certain size (Garry and Thomson, 2009). In the present study, in which TS of 120% of rMT was used, we observed less SICI for small MEPs (Fig. 2). Using moderate suprathreshold TS (110–120% rMT) has yielded the most prominent SICI regardless of excitability (Garry and Thomson, 2009). At ISI of 3 ms, the observed shortening of MEP onset latency and simultaneous decrease of amplitude in APB differs considerably from the “normal” MEP behaviour, where an increase in the number and synchrony of corticospinal volleys leads to shortening of latency and increase of amplitude. A shortening of latency by 1 ms at ISI of 3 ms has been described in the original study by Kujirai et al. (1993). We suggest that at ISI of 3 ms the CS increased the excitability (lowers the threshold) of a spatially rather limited neuronal population in cortex leading to a faster and more synchronous firing of a smaller number of neurons. Non-targeted ADM shows identical behaviour, which strongly suggests that the CS exert its effect either via the spread of tangential cortical fibers, or that the functional cortical representations of APB and ADM overlap sufficiently to react similarly to subthreshold conditioning. At the ISI of 7 ms there was facilitation in amplitude primarily due to already reduced local cortical surround inhibition by CS. Latency remained shortened, and hypothetically, given a stable stimulus delivery, the local neuronal response at 7 ms is optimally synchronized with respect to the size of the reacting population. The ICF was most prominent at the ISI of 13 ms with no local refractory or surround inhibition effects remaining, supported by maximal MEP enhancement and unchanged response latency. Relationship between the amplitude change and latency change could represent the activation of multiple cortical and/or spinal circuits. At an ISI of 28 ms and CS of 90% rMT there was occasional muscle activation observed by EMG indicating significant activation of lower motor neurons in ∼50% of the subjects. These were discarded from the results shown. As the discarded data did not differ from the data analyzed, peripheral involvement can be ruled out. Presumably the ISI of 28 ms exceeds the corticospinal conduction time in most subjects, but it does not allow the direct feedback from muscle spindles or proprioceptive organs to reach the central motor system. Thus, the effects observed are most probably caused by both cortical interneurons and also by some contribution from spinal interneurons. In the light of the present data we cannot speculate

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any further, and the topic of clarifying the mechanisms contributing to longer ISIs requires further studies. The occurrence of SICI and ICF was quite similar in both the optimally targeted APB and the cortically adjacent muscle (ADM), which indicates that PP as a phenomenon is not extremely sensitive to focal targeting. Somewhat surprisingly, ADM showed overall higher responses and stronger ICF at intermediate ISIs when compared to APB. We assume that MT is nearly same in both ADM and APB, but if the MT of ADM was lower, that might explain the small difference observed. Another possible explanation is that APB is actually one of several thenar muscles recorded with surface EMG, whereas ADM is represented by a more focal cortical population which can be activated more synchronously even by a non-optimally targeted pulse. However, the underlying mechanisms of SICI and ICF may not be completely similar in neighbouring representations and warrant further studies. Unintentional coil movement has been suggested to be involved in the well-known large variability in the PP-MEP amplitude, especially for ICF (Hanajima and Ugawa, 2008; Maeda et al., 2002; Orth et al., 2003; Shimizu et al., 1999). TMS is thought to induce a focal excitation circumscribed by a surrounding cortical inhibition (Shimizu et al., 1999; Sohn and Hallett, 2004) and even small shifts in the orientation of the electric field in the brain may cause activation in different neural cell populations, inhibitory or excitatory (Ziemann et al., 1996). With nTMS, it is possible to minimize these small shifts in orientation as well as to control the tilting of the coil during the stimulation (Julkunen et al., 2009). In addition, the produced MEPs with optimal targeting have more stable amplitude and latency with less intra-individual variation as compared to conventional TMS (Julkunen et al., 2009). However, the variability of SICI and ICF in our experiments was still as high as in previous studies without navigation (Boroojerdi et al., 2000b; Cahn et al., 2003; Cicinelli et al., 2000; Maeda et al., 2002; Orth et al., 2003; Wassermann, 2002). One limitation of our study was the low number of stimuli (five) at each ISI which we tried to compensate in the statistical analysis by taking an average on the middle three responses to effectively remove the possible outliers without impairing the value of the data. It is likely that there are still many unknown factors in addition to physiological reasons such as the instable excitability of cortical and spinal motor neurons that cannot be diminished by stabilizing the target of stimulus delivery (Boroojerdi et al., 2000b; Kiers et al., 1993; Rösler et al., 2008; Wassermann, 2002). 5. Conclusions In this study, SICI and ICF were investigated in exploratory manner using TMS. Inhibition was most prominent at ISI of 3 ms and CS of 80% of rMT, whereas CS of 90% of rMT and ISIs of 7 and 13 ms were preferable for facilitation. Latency shortening was observed and appeared stable and independent indicator of both phenomena. The loss of SICI can be detected quite well with the described PP paradigm, which could be used as an indicator of certain motor deficits such as hand dystonia. The occurrence of SICI and ICF was similar in both the optimally targeted APB and the cortically adjacent muscle (ADM) making PP not extremely sensitive to correct targeting. Moreover, the interhemispheric difference, even if statistically significant, had a negligible effect in amount. Hence, PP could be used in characterizing unilateral damage or lateralized motor deficits. Acknowledgements We thank Drs. Selja Vaalto and Nils Danner for the constructive comments on the manuscript. The study has been supported by

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funding from the National Technological Agency of Finland (TEKES) Helsinki, Finland (grant number 893/04), Kuopio University Hospital, Kuopio, Finland (EVO-grant 5041716), The Finnish Cultural Foundation of Northern Savo as well as Emil Aaltonen Foundation, Tampere, Finland. References Awiszus F. TMS and threshold hunting. Suppl Clin Neurophysiol 2003;56:13–23. Berweck S, Walther M, Brodbeck V, Wagner N, Koerte I, Henschel V, et al. Abnormal motor cortex excitability in congenital stroke. Pediatr Res 2008;63(1): 84–8. Boroojerdi B, Battaglia F, Muellbacher W, Cohen LG. Voluntary teeth clenching facilitates human motor system excitability. Clin Neurophysiol 2000a;111: 988–93. Boroojerdi B, Kopylev L, Battaglia F, Facchini S, Ziemann U, Muellbacher W, et al. Reproducibility of intracortical inhibition and facilitation using the paired-pulse paradigm. Muscle Nerve 2000b;23:1594–7. Bütefisch CM, Kleiser R, Seitz RJ. Post-lesional cerebral reorganisation: evidence from functional neuroimaging and transcranial magnetic stimulation. J Physiol Paris 2006;99:437–54. Cahn SD, Herzog AG, Pascual-Leone A. Paired-pulse transcranial magnetic stimulation: effects of hemispheric laterality, gender, and handedness in normal controls. J Clin Neurophysiol 2003;20:371–4. Chen R, Cros D, Curra A, Di Lazzaro V, Lefaucheur JP, Magistris MR, et al. The clinical diagnostic utility of transcranial magnetic stimulation: report of an IFCN committee. Clin Neurophysiol 2008;119:504–32. Chen R, Tam A, Butefisch C, Corwell B, Ziemann U, Rothwell JC, et al. Intracortical inhibition and facilitation in different representations of the human motor cortex. J Neurophysiol 1998;80:2870–81. Cicinelli P, Traversa R, Oliveri M, Palmieri MG, Filippi MM, Pasqualetti P, et al. Intracortical excitatory and inhibitory phenomena to paired transcranial magnetic stimulation in healthy human subjects: differences between the right and left hemisphere. Neurosci Lett 2000;288:171–4. Civardi C, Cavalli A, Naldi P, Varrasi C, Cantello R. Hemispheric asymmetries of cortico-cortical connections in human hand motor areas. Clin Neurophysiol 2000;111:624–9. Claus D, Weis M, Jahnke U, Plewe A, Brunholzl C. Corticospinal conduction studied with magnetic double stimulation in the intact human. J Neurol Sci 1992;111:180–8. Cohen LG, Ziemann U, Chen R, Classen J, Hallett M, Gerloff C, et al. Studies of neuroplasticity with transcranial magnetic stimulation. J Clin Neurophysiol 1998;15:305–24. Daskalakis ZJ, Christensen BK, Fitzgerald PB, Roshan L, Chen R. The mechanisms of interhemispheric inhibition in the human motor cortex. J Physiol 2002;543:317–26. Di Lazzaro V, Restuccia D, Oliviero A, Profice P, Ferrara L, Insola A, et al. Magnetic transcranial stimulation at intensities below active motor threshold activates intracortical inhibitory circuits. Exp Brain Res 1998;119:265–8. Elias LJ, Bryden MP, Bulman-Fleming MB. Footedness is a better predictor than is handedness of emotional lateralization. Neuropsychologia 1998;36:37–43. Ferreri F, Pasqualetti P, Määttä S, Ponzo D, Ferrarelli F, Tononi G, et al. Human brain connectivity during single and paired pulse transcranial magnetic stimulation. NeuroImage 2011;54(1):90–102. Fisher RJ, Nakamura Y, Bestmann S, Rothwell JC, Bostock H. Two phases of intracortical inhibition revealed by transcranial magnetic threshold tracking. Exp Brain Res 2002;143:240–8. Garry MI, Thomson RH. The effect of test TMS intensity on short-interval intracortical inhibition in different excitability states. Exp Brain Res 2009;193:267–74. Hanajima R, Furubayashi T, Iwata NK, Shiio Y, Okabe S, Kanazawa I, et al. Further evidence to support different mechanisms underlying intracortical inhibition of the motor cortex. Exp Brain Res 2003;151:427–34. Hanajima R, Ugawa Y. Paired-pulse measures. In: Wassermann EM, Epstein CM, et al., editors. The Oxford Handbook of Transcranial Stimulation. New York: Oxford University Press; 2008. p. 103–17. Ilic TV, Meintzschel F, Cleff U, Ruge D, Kessler KR, Ziemann U. Short-interval pairedpulse inhibition and facilitation of human motor cortex: the dimension of stimulus intensity. J Physiol 2002;545:153–67. Julkunen P, Säisänen L, Danner N, Niskanen E, Hukkanen T, Mervaala E, et al. Comparison of navigated and non-navigated transcranial magnetic stimulation for motor cortex mapping, motor threshold and motor evoked potentials. NeuroImage 2009;44:790–5. Kapogiannis D, Wassermann EM. Transcranial magnetic stimulation in clinical pharmacology. Cent Nerv Syst Agents Med Chem 2008;8:234–40.

Kiers L, Cros D, Chiappa KH, Fang J. Variability of motor potentials evoked by transcranial magnetic stimulation. Electroencephalogr Clin Neurophysiol 1993;89:415–23. Kossev AR, Siggelkow S, Dengler R, Rollnik JD. Intracortical inhibition and facilitation in paired-pulse transcranial magnetic stimulation: effect of conditioning stimulus intensity on sizes and latencies of motor evoked potentials. J Clin Neurophysiol 2003;20:54–8. Kujirai T, Caramia MD, Rothwell JC, Day BL, Thompson PD, Ferbert A, et al. Corticocortical inhibition in human motor cortex. J Physiol 1993;471:501–19. Liepert J, Bar KJ, Meske U, Weiller C. Motor cortex disinhibition in Alzheimer’s disease. Clin Neurophysiol 2001;112:1436–41. Maeda F, Gangitano M, Thall M, Pascual-Leone A. Inter- and intra-individual variability of paired-pulse curves with transcranial magnetic stimulation (TMS). Clin Neurophysiol 2002;113:376–82. Nakamura H, Kitagawa H, Kawaguchi Y, Tsuji H. Intracortical facilitation and inhibition after transcranial magnetic stimulation in conscious humans. J Physiol 1997;498(Pt 3):817–23. Niskanen E, Julkunen P, Säisänen L, Vanninen R, Karjalainen P, Könönen M. Grouplevel variations in motor representation areas of thenar and anterior tibial muscles: navigated transcranial magnetic stimulation study. Human Bain Mapping 2010;31:1272–80. Orth M, Snijders AH, Rothwell JC. The variability of intracortical inhibition and facilitation. Clin Neurophysiol 2003;114:2362–9. Peurala SH, Muller-Dahlhaus JF, Arai N, Ziemann U. Interference of short-interval intracortical inhibition (SICI) and short-interval intracortical facilitation (SICF). Clin Neurophysiol 2008;119:2291–7. Reis J, Swayne OB, Vandermeeren Y, Camus M, Dimyan MA, Harris-Love M, et al. Contribution of transcranial magnetic stimulation to the understanding of cortical mechanisms involved in motor control. J Physiol 2008;586:325–51. Ridding MC, Inzelberg R, Rothwell JC. Changes in excitability of motor cortical circuitry in patients with Parkinson’s disease. Ann Neurol 1995;37:181–8. Rosenkranz K, Williamon A, Rothwell JC. Motorcortical excitability and synaptic plasticity is enhanced in professional musicians. J Neurosci 2007;27:5200–6. Roshan L, Paradiso GO, Chen R. Two phases of short-interval intracortical inhibition. Exp Brain Res 2003;151:330–7. Rossini PM, Barker AT, Berardelli A, Caramia MD, Caruso G, Cracco RQ, et al. Noninvasive electrical and magnetic stimulation of the brain, spinal cord and roots: basic principles and procedures for routine clinical application. Report of an IFCN committee. Electroencephalogr Clin Neurophysiol 1994;91:79–92. Ruohonen J, Karhu J. Navigated transcranial magnetic stimulation. Neurophysiol Clin 2010;40:7–17. Rösler KM, Roth DM, Magistris MR. Trial-to-trial size variability of motor-evoked potentials. A study using the triple stimulation technique. Exp Brain Res 2008;187:51–9. Shimizu T, Filippi MM, Palmieri MG, Oliveri M, Vernieri F, Pasqualetti P, et al. Modulation of intracortical excitability for different muscles in the upper extremity: paired magnetic stimulation study with focal versus non-focal coils. Clin Neurophysiol 1999;110:575–81. Shirota Y, Hamada M, Terao Y, Matsumoto H, Ohminami S, Furubayashi T, et al. Influence of short-interval intracortical inhibition on short-interval intracortical facilitation in human primary motor cortex. J Neurophysiol 2010;104(3):1382–91. Sohn YH, Hallett M. Surround inhibition in human motor system. Exp Brain Res 2004;158:397–404. Säisänen L, Julkunen P, Niskanen E, Danner N, Hukkanen T, Lohioja T, et al. Motor potentials evoked by navigated transcranial magnetic stimulation in healthy subjects. J Clin Neurophysiol 2008:367–72. Vucic S, Cheah BC, Krishnan AV, Burke D, Kiernan MC. The effects of alterations in conditioning stimulus intensity on short interval intracortical inhibition. Brain Res 2009;1273:39–47. Wagle-Shukla A, Ni Z, Gunraj CA, Bahl N, Chen R. Effects of short interval intracortical inhibition and intracortical facilitation on short interval intracortical facilitation in human primary motor cortex. J Physiol 2009;587:5665–78. Wassermann EM. Variation in the response to transcranial magnetic brain stimulation in the general population. Clin Neurophysiol 2002;113:1165–71. Yousry TA, Schmid UD, Alkadhi H, Schmidt D, Peraud A, Buettner A, et al. Localization of the motor hand area to a knob on the precentral gyrus. A new landmark. Brain 1997;120(Pt 1):141–57. Ziemann U. Intracortical inhibition and facilitation in the conventional paired TMS paradigm. Electroencephalogr Clin Neurophysiol Suppl 1999;51:127–36. Ziemann U. TMS and drugs. Clin Neurophysiol 2004;115:1717–29. Ziemann U, Rothwell JC, Ridding MC. Interaction between intracortical inhibition and facilitation in human motor cortex. J Physiol 1996;496(Pt 3):873–81. Ziemann U, Tergau F, Wassermann EM, Wischer S, Hildebrandt J, Paulus W. Demonstration of facilitatory I wave interaction in the human motor cortex by paired transcranial magnetic stimulation. J Physiol 1998;511(Pt 1):181–90.