www.elsevier.com/locate/ynimg
NeuroImage 28 (2005) 22 – 29
BOLD MRI responses to repetitive TMS over human
dorsal premotor cortex
Sven Bestmann,a,b,* Jurgen Baudewig,a Hartwig R. Siebner,c
John C. Rothwell,b and Jens Frahma
aBiomedizinische NMR Forschungs GmbH am Max-Planck-Institut fur Biophysikalische Chemie, 37070 Gottingen, GermanybSobell Department of Motor Neuroscience and Movement Disorders, Institute of Neurology, University College of London, UKcInstitute of Neurology, University of Kiel, Germany
Received 20 June 2004; revised 20 April 2005; accepted 20 May 2005
Available online 5 July 2005
Functional magnetic resonance imaging (fMRI) studies in humans have
hitherto failed to demonstrate activity changes in the direct vicinity of
transcranialmagnetic stimulation (TMS) that cannot be attributed to re-
afferent somatosensory feedback or a spread of excitation. In order to
investigate the underlying activity changes at the site of stimulation as
well as in remote connected regions, we applied short trains of high-
intensity (110% of resting motor threshold) and low-intensity (90% of
activemotor threshold) repetitive TMS (rTMS; 3 Hz, 10 s duration) over
the presumed location of the left dorsal premotor cortex (PMd) during
fMRI. Signal increases in the direct vicinity of the stimulated PMd were
observed during rTMS at 110% RMT. However, positive BOLD MRI
responses were observed with rTMS at both 90% and 110% RMT in
connected brain regions such as right PMd, bilateral PMv, supplemen-
tary motor area, somatosensory cortex, cingulate motor area, left
posterior temporal lobe, cerebellum, and caudate nucleus. Responses
were generally smaller during low-intensity rTMS. The results indicate
that short trains of TMS can modify local hemodynamics in the absence
of overt motor responses. In addition, premotor rTMS cannot only
effectively stimulate cortico-cortical but also cortico-subcortical con-
nections even at low stimulation intensities.
D 2005 Elsevier Inc. All rights reserved.
Keywords: fMRI; EMG; SMA; BOLD; Echo-planar imaging; Motor cortex
Introduction
A number of recent combined transcranial magnetic stimulation
(TMS) and functional magnetic resonance imaging (fMRI) studies
have confirmed that TMS leads to changes in neural activity in
structures beyond the site of stimulation (Bohning et al., 1998,
1053-8119/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.neuroimage.2005.05.027
* Corresponding author. Biomedizinische NMR Forschungs GmbH am
Max-Planck-Institut fur Biophysikalische Chemie, 37070 Gottingen,
Germany. Fax: +49 551 201 1307.
E-mail address: [email protected] (S. Bestmann).
Available online on ScienceDirect (www.sciencedirect.com).
1999, 2000b; Bestmann et al., 2004). Although similar effects have
been observed with positron emission tomography (PET), the
ability to perform similar experiments in fMRI allows us safely to
make repeated studies on the same individuals at higher temporal
and spatial resolution than can be obtained in PET (Bohning et al.,
1998, 1999, 2000b; Shastri et al., 1999; Baudewig et al., 2001;
Nahas et al., 2001; Bestmann et al., 2004). However, despite their
advantages, combined TMS-fMRI studies have produced results
that have led to one puzzling question: what happens at the site of
stimulation itself? A problem has been that most studies have used
TMS over motor cortex, and stimulation here can lead to
contralateral movements and sensory feedback that additionally
contribute to BOLD MRI signal changes. In fact, stimulation of
motor cortex at sub-movement threshold intensities fails to change
BOLD activity under the TMS coil, even though changes can be
seen at other distant connected sites (Bohning et al., 1998, 1999,
2000b; Baudewig et al., 2001; Kemna and Gembris, 2003;
Bestmann et al., 2004). Interestingly, no significant changes in
activity have been reported at sites of stimulation in the parietal and
premotor cortex in a recent repetitive TMS (rTMS) study even
when using intensities well above the threshold needed to activate
the primary motor cortex (Kemna and Gembris, 2003). One
possible explanation is that the threshold for motor cortex
stimulation is not the same as the threshold in non-motor regions
(Stewart et al., 2001). Thus, the absence of significant local activity
changes in these experiments may simply reflect the fact that the
intensity or duration was not sufficient to cause significant BOLD
MRI response changes.
Here, we performed TMS-fMRI over the presumed location of
the dorsal premotor cortex in order to investigate the occurrence of
activity changes in the direct vicinity of the stimulation site as well as
remote brain regions. This stimulation site has an advantage over
motor cortex stimulation in that it does not provoke movements of
contralateral muscles and therefore avoids the confounding BOLD
signals that result from reafferent feedback. Although the precise site
of stimulation with the TMS coil is not well defined, the effects of
S. Bestmann et al. / NeuroImage 28 (2005) 22–29 23
TMS over this site differ from those seen after stimulation over the
motor cortex in both their physiological effects on corticocortical
connectivity (Civardi et al., 2001; Mochizuki et al., 2004; Rizzo et
al., 2004) as well as their metabolic consequences as recorded with
PET (Chouinard et al., 2003; Siebner et al., 2003). For these reasons,
we have referred to the site as dorsal premotor cortex since it is
anterior and slightly medial to the TMS ‘‘hot spot’’ for provoking
EMG activity in contralateral hand muscle.
Using the premotor site as a model, we asked (a) whether TMS-
fMRI is capable of visualizing activity changes in the direct
vicinity of the TMS probe and (b) whether secondary motor and
non-motor regions that are connected to PMd show activity
changes during TMS-fMRI. We show that rTMS over premotor
cortex activates a large network of remote interconnected cortical
and subcortical motor regions. The results furthermore demonstrate
for the first time that short TMS trains modulate local hemody-
namic responses in the absence of overt muscle movements.
Materials and methods
Subjects
We studied nine right-handed healthy subjects without any
previous personal or family neuropsychiatric or neurological
history (mean age 29 years, range 25–42 years; six female) after
receiving written informed consent. Examinations conformed to
the standards laid down by the Declaration of Helsinki and local
ethics board approval.
Experimental procedures
In each of three experimental conditions, eight stimulation
epochs (9.96 s) alternated with resting periods (23.24 s). Subjects
were instructed to keep their eyes closed and to relax their hands
while repetitive TMS was applied at 3 Hz using two different
intensities: 110% of individual resting motor threshold (RMT) and
90% of individual active motor threshold (AMT) (mean: 92%
(73%) of the maximal output of the stimulator, range: 82–100%
(65–81%). The high stimulation intensity values are caused by the
introduction of the long connecting cable to the TMS coil allowing
it to be used within the scanner. This increases the resistance and
inductance of the circuit so that higher intensities of stimulator
output are required to generate the same maximum current flow.
Using the same protocol timings, participants dorsiflexed the right
index finger in order to locate areas of increased BOLD MRI signal
related to focal hand movement. The time of each movement was
cued by applying (ineffective) rTMS at 15% of stimulator output
(mean: 21% of AMT, range: 19–23%). The order of experimental
conditions was pseudo-randomized in order to avoid the same
TMS condition occurring in successive scans (five subjects started
with suprathreshold TMS). The three experimental runs were
separated by 5 min to reduce carry-over effects of stimulation. The
number and intensity of TMS pulses conformed to presently
available safety guidelines for TMS (Wassermann, 1998).
Magnetic resonance imaging
MRI was performed at 2.9 T (Siemens Trio, Erlangen,
Germany) using a standard transmit-receive head-coil. Anatomical
images were acquired using a short-echo time 3D FLASH
sequence (TR/TE = 11/4.92 ms, flip angle 15-, 1 mm isotropic
resolution) covering the whole head. Functional MRI was
conducted using a T2*-weighted single-shot gradient-echo EPI
sequence (frequency-selective fat suppression, TR/TE = 3320/36
ms, flip angle 70-, 2 � 2 mm2 resolution, 4 mm section thickness).
Twenty oblique brain sections covered a region from the primary
and secondary motor cortex down to the basal ganglia and the
thalamus. Each fMRI run lasted 4 min 59 s, corresponding to 90
volumes including periods for signal equilibration.
Transcranial magnetic stimulation and electromyographic
recordings
TMS was conducted using a non-ferromagnetic figure-of-eight
coil (two windings of ten turns each; inner winding diameter 53
mm; distance between outer coil surface and windings: 2–3 mm
(variation due to manufacturing tolerance); coil inductance includ-
ing cable: 20 AH; maximal current at 100% stimulator output:
approximately 5 kA). The coil was connected to a Magstim Rapid
stimulator (The Magstim Company, Wales, UK) outside the
radiofrequency-shielded cabin via an 8 m cable inserted through
a filter tube. This ensured that no other material other than the
MRI-compatible TMS coil and the connecting cable could be
introduced into the scanner room. The TMS coil was mechanically
strengthened for the requirements at 3 T by inclusion of an
appropriately shaped 8 mm plastic former on the reverse side of the
coil which was attached to the coil by high strength fiber tape (The
Magstim Company). Each subject wore earplugs and headphones
to reduce acoustic noise from the discharging TMS coil.
Biphasic electrical pulses (approximate rise time based on full
coil inductance including connecting cable and coil windings:
approximately 90 As with a duration of 250 As) were applied with
an initial anteroposterior direction of current flow. TMS-fMRI was
synchronized using Presentation 0.51 software (Neurobehavioral
Systems, Inc., San Francisco, USA).
Before scanning, the optimal position of the coil for evoking
movements of the contralateral hand (referred to as motor hot spot)
was determined. Individual motor thresholds were then obtained
with single TMS pulses. RMT was defined as the minimum
intensity that evoked a muscle twitch in 5 out of 10 trials that was
clearly observable to two of the experimenters (S.B., J.B.). The
minimum intensity that caused an observable muscle twitch during
10% of maximum voluntary contraction in 5 out of 10 trials was
defined as AMT.
The site for premotor stimulation was defined with reference to
the motor hot spot according to previously published procedures
(Schluter et al., 1998, 1999; Johansen-Berg et al., 2002). The coil
was moved 2 cm anterior and 1 cm medial and fixed using a
custom-made adjustable coil holder (Polyetheretherketon, PEEK)
mounted onto the MRI head coil. In two subjects, suprathreshold
TMS at the target site evoked electromyographic (EMG) responses
in the relaxed right first dorsal interosseous (FDI) muscle, and the
coil was moved 0.5 and 1 cm anterior to the target site,
respectively. This was motivated by the fact that the presumed
area of stimulation along the junction of the TMS coil wings spans
a region of about 4 cm (Barker, 1999) and thus the motor hot spot
was still within the target region of the PMd. Moreover, previous
studies have shown a good correspondence of scalp positions
between 2 and 3 cm anterior to the motor hot spot and the
underlying dorsal premotor complex (Johansen-Berg et al., 2002;
Chen et al., 2003; Siebner et al., 2003; Oxley et al., 2004).
S. Bestmann et al. / NeuroImage 28 (2005) 22–2924
Consequently, we aimed to minimize spread of excitation and overt
muscle movements while at the same time retaining the TMS probe
over the presumed dorsal premotor region.
During fMRI, EMG responses from the right FDI muscle were
continuously monitored to assess a possible spread of excitation
into the adjacent M1. Surface EMG was recorded with non-
ferromagnetic sintered Ag/AgCl surface electrodes (9 mm diam-
eter) using a belly-tendon montage. The electrodes were connected
to a BrainAmp MRI-compatible EEG amplifier (Brain Products,
Munich, Germany). EMG data were recorded at a sampling rate of
2000 Hz, rectified, and low-pass filtered at 50 Hz using Brain
Vision 1.0 (Brain Products, Munich, Germany). Following the
removal of MRI gradient artefacts, the residual EMG allowed for a
qualitative detection of electromyographic activity. Recordings
from each experimental run were normalized to the mean EMG
amplitude and paired t tests with Bonferroni correction for multiple
comparisons were used to compare EMG levels during TMS and
resting epochs.
Combined TMS-fMRI
Combined TMS-fMRI was accomplished as described previ-
ously (Bohning et al., 1998; Shastri et al., 1999; Bestmann et al.,
2003a, 2004). Functional images were acquired every 166 ms,
while each image acquisition lasted for 91 ms and TMS pulses
were applied immediately afterwards. The chosen stimulation
frequency of 3 Hz (every 332 ms) provided waiting periods
between TMS pulses and subsequent image acquisitions of 75 ms
which allowed for unperturbed fMRI as confirmed in pilot
experiments.
Data analysis
Image processing and statistical analyses were carried out using
BrainVoyager 2000 (Brain Innovation, Maastricht, The Nether-
lands). Realignment, intensity normalization, spatial smoothing
and linear drift removal was performed prior to statistical analysis
following procedures described elsewhere (Goebel et al., 1998).
2D functional slice-time data were co-registered with anatomical
T1-weighted images from the same session. Functional images
were transformed into 3 mm isotropic resolution. Individual pre-
processed volume time courses were analyzed using the general
linear model (GLM) with stimulation or movement epochs as the
effects of interest. Multi-subject analysis was conducted using a
fixed effects model to test for significant changes in BOLD MRI
signal during each experimental condition at a group level (P <
0.01 adjusted for multiple comparisons). Coordinates of the center
of gravity of activation clusters were determined with reference to
the MNI template (Montreal Neurological Institute) in stereotaxic
space (Talairach and Tournoux, 1988). EMG recordings from each
session were normalized to the mean EMG amplitude and mean
normalized EMG levels during TMS and resting epochs were
compared using paired samples t tests.
Fig. 1. Electromyographic (EMG) recordings of (a) voluntary finger tapping
(right hand) and (b) suprathreshold rTMS of the left PMd from a
representative subject recorded during simultaneous fMRI. Stimulation
epochs (boxes) were time-locked averaged to the onset of stimulation.
Absence of EMG activity in the contralateral first dorsal interosseous (FDI)
muscle during suprathreshold rTMS indicated that stimulation at 110%
resting motor threshold did not spread into the primary motor hand area.
Results
None of the subjects reported any side effects from the
experimental procedure when asked immediately after the experi-
ment. No additional interrogation was conducted subsequently.
Compared with resting epochs, no significant EMG activity was
observed during suprathreshold rTMS in any of the subjects
(paired samples t test, P = 0.24, df = 8), whereas voluntary finger
movement epochs evoked clear EMG responses (P < 0.01, df = 8).
Fig. 1 presents EMG recordings of a representative subject.
Repetitive TMS at 110% RMT produced a localized BOLD
MRI signal increase in the left PMd (mean coordinates of activated
cluster: x = �40, y = �11, z = 54; see Fig. 2 and Table 1).
Additional activity increases were found in the homologous PMd,
the bilateral ventral premotor region, the supplementary motor area
(SMA) in the medial aspect of the superior frontal gyrus, and the
putative cingulate motor area. In contrast, no significant changes
were found in the left primary sensorimotor cortex. Repetitive
TMS at 110% RMT was also associated with increases in BOLD
MRI signal in the left posterior middle temporal gyrus, and large
parts of the putative bilateral auditory cortex including the superior
temporal plane, superior temporal gyrus, and planum temporale,
extending dorsally into putative SII (Fig. 2, Table 1). Subcortically,
bilateral activity was found in the middle part of the caudate
nucleus, as well as in the thalamus of the left hemisphere and the
bilateral inferior colliculi. In addition, focal BOLD MRI responses
were found in the medio-dorsal cerebellum, with a right-hemi-
spheric preponderance (Table 1).
As shown in Fig. 3 and Table 2, there was a close spatial
correspondence between brain regions activated during voluntary
finger movement and rTMS of the left PMd. This was confirmed
by a conjunction analysis which tested for brain regions showing
an increase in BOLD MRI signal during both voluntary finger
movement and rTMS at 110% RMT (Table 2). The results
demonstrate that activations common to both experimental con-
ditions comprised large parts of motor system known to be
involved in manual motor control except for the left M1 which was
only found to be activated during voluntary movement.
The brain regions that revealed an increase in BOLD MRI
signal during subthreshold rTMS are listed in Table 3. In contrast
to suprathreshold stimulation, no significant response was
observed in the targeted presumed PMd (Fig. 5a). BOLD MRI
signal increases in distant brain regions to low-intensity rTMS
were highly co-localized with changes during stimulation at 110%
RMT. However, the extent of activation clusters as well as peak
Table 1
Activity evoked by suprathreshold rTMS over the left PMd
Anatomical/functional location xa ya za t valueb Volumec
L premotor (dorsal) �40 �11 54 8.24 438
L premotor (ventral) �49 2 17 10.29 1339
L auditory cortexd �45 �25 15 16.42 7836
L middle temporal gyrus �50 �52 3 9.26 1299
L thalamus �14 �17 8 6.04 150
L caudate nucleus �11 �4 18 6.72 68
L putamen �27 �11 3 5.82 276
L cerebellar hemisphere �6 �59 �20 6.99 355
L inferior colliculus �3 �20 �2 7.00 56
BL medial superior frontal
gyrus (SMA)
3 �5 55 8.73 898
BL cingulate gyrus 3 4 44 9.26 908
R premotor (dorsal) 43 �3 49 8.92 367
R premotor (dorsal) 42 7 26 7.64 238
R premotor (ventral) 54 0 13 9.14 1127
R auditory cortexd 50 �22 15 15.72 7311
R thalamus 12 �14 8 6.97 172
R caudate nucleus 12 0 14 7.42 630
R inferior colliculus 6 �25 �6 7.80 93
R cerebellar hemisphere 5 55 �15 8.38 701
a Coordinates correspond to center of gravity of respective activation
clusters.b Peak activation within cluster with P < 0.01, corrected.c Rescaled to voxel size 1 � 1 � 1 mm.d Including superior temporal gyrus, ventral part of parietal operculum
and planum temporale. L: left, R: right, BL: bilateral.Fig. 2. BOLD MRI responses to suprathreshold rTMS of left premotor
cortex (group analysis, n = 9, P < 0.01, corrected). (a) Sagittal (x = �40),
coronal ( y = �11), and transverse (z = 55) view of activity in the left PMd.
(b) Six transverse sections showing activity changes in the cingulate gyrus,
PMv, auditory cortex, caudate nucleus, left posterior temporal lobe, medial
geniculate nucleus, and cerebellum with coordinates indicated. Activation
maps are projected onto a template brain (Montreal Neurological Institute,
MNI). L: left, R: right.
S. Bestmann et al. / NeuroImage 28 (2005) 22–29 25
activity within activation clusters were markedly reduced during
low-intensity rTMS (Tables 1, 3). A direct comparison revealed
that stimulation at 110% RMT was associated with a stronger
BOLD MRI signal at the site of stimulation and in distant brain
regions than low-intensity rTMS, including lateral and mesial
premotor regions, left posterior temporal gyrus, and the thalamus
bilaterally (Fig. 4, Table 4). Neither high- nor low-intensity rTMS
evoked significant BOLD MRI signal changes in the caudally
adjacent M1 hand area (Fig. 5b).
Fig. 3. Brain regions with BOLD MRI responses to both suprathreshold
rTMS of left premotor cortex and voluntary finger movement of the right
hand (group analysis, n = 9; P < 0.01, corrected). The data are projected
onto a left-hemispheric 3D surface reconstruction (MNI) and reveal
enhanced activity in the stimulated left PMd (red circle). Additional
activity in the left hemisphere was evoked in PMv, SMA, cingulate gyrus,
auditory cortices, and left posterior middle temporal gyrus.
Discussion
The present study provides evidence that suprathreshold rTMS
over the left premotor cortex is able to elicit BOLD MRI signal
increases in the direct vicinity of the targeted region, as well as in a
range of cortical and subcortical distant brain regions. In contrast,
stimulation at 90% AMT increased activity in connected cortical
and subcortical areas but failed to induce significant BOLD MRI
changes in the directly targeted premotor region. Further ROI
analysis confirmed that no consistent change in average BOLD
MRI signal occurred under the probe in the low-intensity
condition. While the spatial pattern of distant activity changes
was comparable for both intensities, these were stronger during
rTMS at 110% RMT as compared to subthreshold stimulation.
Local effects of premotor rTMS
Previous studies reported a local increase in BOLD MRI signal
at the site of stimulation only when rTMS was applied to M1 at
intensities suprathreshold for evoking a contralateral muscle twitch
(Bohning et al., 1998, 1999, 2000a,b; Baudewig et al., 2001;
Kemna and Gembris, 2003; Bestmann et al., 2003b, 2004).
However, these effects most likely had confounding contributions
from somatosensory feedback caused by the contralateral muscle
Fig. 4. Comparison of BOLD MRI responses to supra- and subthreshold
rTMS over left premotor cortex (group analysis, n = 9; P < 0.01, corrected)
in a sagittal (a), coronal (b), and two transverse (c, d) sections of a reference
brain (MNI) with Talairach coordinates indicated. Suprathreshold rTMS
evoked significantly stronger activity changes than subthreshold rTMS at
the stimulated left PMd (red circle), the right PMd, SMA, cingulate gyrus,
right cerebellum, bilateral superior temporal plane (putative auditory cortex
and SII), bilateral caudate nucleus, and left posterior middle temporal gyrus.
Table 4
Differences in activity between supra- and subthreshold rTMS over left
Table 2
Conjunction analysis finger tapping-suprathreshold rTMS
Anatomical/functional location xa ya za t valueb Volumec
L premotor (ventral) �50 0 12 9.21 1047
L auditory cortexd �45 �27 16 13.93 6370
L middle temporal gyrus �50 �53 3 9.26 1014
L thalamus �12 �17 5 5.89 122
L putamen �26 �11 3 6.12 180
L inferior colliculus �5 �19 �3 6.47 119
BL medial superior frontal
gyrus (SMA)
2 �5 53 8.72 727
BL cingulate gyrus 3 5 44 8.39 679
R premotor (ventral) 53 1 14 9.14 620
R auditory cortexd 53 �22 15 14.11 5308
R caudate nucleus 13 �2 15 6.86 200
R cerebellar hemisphere 5 �55 �15 8.38 588
a Coordinates correspond to center of gravity of respective activation
clusters.b Peak activation within cluster with P < 0.01, corrected.c Rescaled to voxel size 1 � 1 � 1 mm.d Including superior temporal gyrus, ventral part of parietal operculum
and planum temporale. L: left, R: right, BL: bilateral.
S. Bestmann et al. / NeuroImage 28 (2005) 22–2926
movements. In the present study, short trains of rTMS at 110%
RMT induced consistent BOLD MRI signal increases in the
stimulated left PMd. This shows for the first time that rTMS
evokes a local hemodynamic response that can now be visualized
by concurrent fMRI. Our simultaneous EMG recordings indicate
that movement-evoked sensory feedback was not the source of
these effects.
Using suprathreshold rTMS, previous prefrontal rTMS-fMRI
studies have failed to provoke significant local BOLD MRI signal
changes with pulse trains of 1 s duration at 10 Hz (Baudewig et al.,
2001) or 4 Hz (Kemna and Gembris, 2003), respectively. This
suggests that not only the high intensity but also the total number
of pulses (i.e., the length of the rTMS train) is critical for inducing
significant activity changes (as indexed by the BOLD MRI signal).
A similar conclusion was reached in several previous PET studies
(Paus et al., 1998; Speer et al., 2003a,b). Dose-dependent effects of
TMS have also been found in the global mean field amplitude,
taken as an index of the overall brain response due to TMS
(Komssi et al., 2004).
Table 3
Activity evoked by subthreshold rTMS over the left PMd
Anatomical/functional location xa ya za t valueb Volumec
L premotor (ventral) �52 �3 15 7.30 811
L auditory cortexd �47 �23 14 12.86 5619
L middle temporal gyrus �48 �53 4 7.63 251
L thalamus �19 �15 4 6.03 60
BL medial superior frontal
gyrus (SMA)
�1 �6 52 6.02 75
BL cingulate gyrus 3 6 43 6.23 231
R premotor (ventral) 53 �1 11 7.79 575
R auditory cortexd 49 �26 16 12.29 5709
R thalamus 12 �16 9 6.63 103
a Coordinates correspond to center of gravity of respective activation
clusters.b Peak activation within cluster with P < 0.01, corrected.c Rescaled to voxel size 1 � 1 � 1 mm.d Including superior temporal gyrus, ventral part of parietal operculum
and planum temporale. L: left, R: right, BL: bilateral.
Evidence from electrophysiological studies of stimulation over
M1 suggests that there is a progressive increase in the
excitability of local circuits during rTMS especially during
stimulation at frequencies above 1 Hz (Pascual-Leone et al.,
1994; Wu et al., 2000). This may cause a progressive increase of
neural activity and subsequent positive BOLD MRI changes.
PMd
Anatomical/functional location xa ya za t valueb Volumec
L premotor (dorsal) �37 �10 53 5.08 157
L premotor (ventral) �51 5 12 5.52 530
L posterior temporal lobe �50 �51 3 4.96 485
L caudate nucleus �10 �5 19 4.14 55
L auditory cortexd �48 �24 16 5.30 532
BL medial superior frontal
gyrus (SMA)
3 �5 55 6.53 297
BL cingulate gyrus 2 5 47 5.40 319
R premotor (dorsal) 43 �8 52 6.79 130
R premotor (ventral) 41 7 26 5.80 130
R caudate nucleus 13 0 15 4.63 414
R auditory cortexd 51 �20 14 5.72 411
R cerebellar hemisphere 5 �55 �14 5.61 336
a Coordinates correspond to center of gravity of respective activation
clusters.b Peak activation within cluster with P < 0.01, corrected.c Rescaled to voxel size 1 � 1 � 1 mm.d Including superior temporal gyrus, ventral part of parietal operculum
and planum temporale. L: left, R: right, BL: bilateral.
Fig. 5. BOLD MRI signal intensity time courses (mean T SEM, n = 9) in (a) the left PMd and (b) the left M1 as determined from a priori anatomically defined
regions-of-interest (Bestmann et al., 2004). Voluntary finger movement (solid line) and suprathreshold rTMS (dashed line) evoked responses in the stimulated
left premotor cortex, while no significant signal changes were found during subthreshold rTMS (dotted line). In the left M1 caudal to the site of rTMS, only
finger movements resulted in significant activity changes.
S. Bestmann et al. / NeuroImage 28 (2005) 22–29 27
Another possibility is that with increasing numbers of TMS
pulses or higher intensities of individual pulses, there is a gradual
accumulation of oxygen debt that eventually triggers subsequent
blood flow changes. If this were the case, a certain amount of
TMS-evoked activity within a certain time window would be
required to trigger BOLD MRI signal changes. Both views
would be consistent with the lack of significant local BOLD
MRI response changes during short subthreshold TMS applica-
tions (Bohning et al., 2000b; Baudewig et al., 2001). The
absence of significant BOLD MRI changes during subthreshold
rTMS, however, does not exclude the possibility that stimulation
led to changes in local intracortical activity (Sanger et al., 2001).
However, this would mean that combined TMS-fMRI may be
blind to subtle and short-lasting activity changes within intra-
cortical circuits that can be readily detected using surface EMG
or cervical epidural recordings.
Distant effects of premotor rTMS
Remote cortical and subcortical activity changes occurred in
secondary motor areas including the contralateral PMd, the ventral
premotor cortex, cingulate, and SMA bilaterally, and subcortically,
the thalamus, caudate nucleus, and putamen. The nature of the
remote effect of TMS is not well understood. The presumed net
facilitatory effect on overall (inhibitory and excitatory) neural
activity in remote regions may be produced by transsynaptic or
direct (orthodromic or antidromic) activation of cortico-cortical, or
cortico-subcortical neurons. While a combination of these is likely
to account for remote activity changes at sites with relatively direct
connections, antidromic activation is a less likely candidate for a
polysynaptic propagation of activity to regions such as the
contralateral thalamus.
Activity increases in the homologous right PMd during
suprathreshold rTMS could be caused by direct activation of
callosal connections (Marconi et al., 2003) by each TMS pulse.
However, direct activation of transcallosal fibers during subthres-
hold stimulation is less likely given that these fibers have a smaller
diameter than the corticospinal fibers from M1 that are activated at
suprathreshold stimulation intensities. It seems more likely that
subthreshold rTMS changed the ongoing pattern of activity in
connections from the PMd to remote target regions by altering the
activity in local circuits intrinsic to PMd itself.
Notably, no significant activity changes were found in the
adjacent left M1/S1. This contrasts with recent studies that have
shown physiological effects on motor cortex excitability after
single pulse TMS (Civardi et al., 2001) and metabolic as well as
physiological effects on M1/S1 after long periods of premotor
rTMS (Chouinard et al., 2003; Siebner et al., 2003). The different
results in the present experiment may be due to a combination of
the fact that we used a different frequency of rTMS than had been
used in previous studies as well as a smaller number of pulses.
Since the pattern of excitatory and inhibitory activity produced by
rTMS is critically dependent on these parameters, it may have been
that we evoked activation of M1/S1 that resulted in no net change
in metabolic activity.
As TMS generates auditory and vibrotactile stimulation on the
scalp, this raises the question whether activity changes are
mediated by these factors rather than direct effective cortical
stimulation and its propagation into remote regions. In the present
experiments, the comparison of the two effective TMS intensities
revealed a number of secondary motor regions, including
contralateral PMd, PMv, SMA, cingulate motor region, and the
caudate nucleus, that were activated more strongly during supra-
threshold rTMS. In contrast, auditory activation was only margin-
ally larger during suprathreshold rTMS. The finding that there was
extensive overlap in auditory cortices even during finger tapping
(at which TMS was applied at 15% of stimulator output) suggests
that these activations are not due to the cortical stimulation itself,
but rather due to the auditory stimulation.
Notwithstanding the secondary activity changes related to
somatosensory and auditory stimulation, TMS applications to the
motor system target distinct cortical and subcortical circuits that
reflect the known anatomical relationship between such areas.
This means that it is possible to test the effective connectivity of
a variety of brain regions by using TMS to provide a controlled
input into a cortical circuit(s). Although the importance of
stimulus intensity on local and distal BOLD signal changes
needs to be explored further across a wide range of stimulus
intensities, the present results are proof of principle that combined
TMS-fMRI over non-primary motor regions can be used to
monitor functional connectivity in cortical circuits. In the future,
we hope to be able to apply the method to explore how these
systems react to acute and chronic perturbation of function in
healthy and disease.
S. Bestmann et al. / NeuroImage 28 (2005) 22–2928
Methodological considerations
Several factors suggest that the PMd was the target of our
TMS pulses. First, the lack of EMG activity makes a contribution
from the caudally adjoining M1 unlikely. Second, stimulation of
the nearby frontal eye field (Paus et al., 1997) or the adjacent
anterior dorsolateral prefrontal cortex (Nahas et al., 2001) has not
yielded prefrontal motor regions in a comparable way. Third, the
activation center in left caudal PMd during suprathreshold rTMS
(x = �40, y = �11, z = 54) was 12 mm anterior to the activation
center in M1/S1 during finger tapping (x = 33, y = �23, z = 55).
This corresponds well to the average distance between the PMd
and M1 (Picard and Strick, 2001). This pattern was preserved
when the two subjects with a more anterior stimulation site were
excluded from the analysis. Previous studies have shown that
when the TMS coil is over the motor hot spot, the center of the
junction region lies 5–10 mm from the site of maximum BOLD
signal change during finger movement (Herwig et al., 2002; Lotze
et al., 2003). Thus, movement of the TMS probe 2–2.5 cm
anterior to the motor hot-spot would be compatible the idea that
the BOLD MRI signal change 12 mm anterior to the M1/S1
activation center was caused by TMS pulses over PMd. However,
it is important to emphasize that we cannot exclude the possibility
of additional stimulation of more prefrontal regions. Nevertheless,
given that micro- and macroanatomical differences between brain
regions make a determination of the exact site of the maximal
induced current and most effective stimulation difficult, activity
changes in the direct vicinity of stimulation may provide an
indication of the most effectively targeted cortical region. We
conclude that in the present study movement of the TMS coil 2–
2.5 cm anterior to the motor hotspot effectively influenced activity
in the dorsal premotor cortex, rather than the anterior dorsolateral
prefrontal cortex.
Due to technical restrictions we used a stimulation frequency
between the commonly applied frequencies of 1 Hz and 5 Hz. It is
therefore difficult to interpret the results directly with regard to the
predominant inhibitory (1 Hz) and excitatory (5 Hz) effects of
rTMS. However, our results clearly demonstrate that rTMS at a
relatively low frequency can already exert significant influence on
both local and remote brain regions. In the future, the direct
comparison of different stimulation frequencies may provide more
detailed information regarding the contribution of inhibitory and
excitatory circuits to such activity changes.
Acknowledgments
SB (DFG GK-GRK 632/1-00) and HRS (DFG SI 738/1) are
grateful for financial support from the Deutsche Forschungsge-
meinschaft. The authors would like to thank John R Hernshey and
Anders A Baumann for assistance and are indebted to Anthony
Thomas and Stefan Cohrs for technical support.
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