Journal of Neuroscience Methods 172 (2008) 270–276

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Journal of Neuroscience Methods

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Navigated TMS combined with EEG in mild cognitive impairment and

Alzheimer’s disease: A pilot study
Petro Julkunen a,∗ , Anne M. Jauhiainen b , Susanna Westerén-Punnonen a , Eriikka Pirinen a ,
Hilkka Soininen b,c , Mervi Könönen a,d , Ari Pääkkönen a , Sara Määttä a , Jari Karhu a,e
a
Department of Clinical Neurophysiology, Kuopio University Hospital, POB 1777,
FI-70211 Kuopio, Finland
b
Institute of Clinical Medicine, Unit of Neurology, University of Kuopio, POB 1627, FI-70211 Kuopio, Finland
c
Department of Neurology, Kuopio University Hospital, POB 1777, FI-70211 Kuopio, Finland
d
Department of Clinical Radiology, Kuopio University Hospital, POB 1777, FI-70211 Kuopio, Finland
e
Nexstim Ltd., Elimäenkatu 9 B, FI-00510 Helsinki, Finland

a r t i c l e i n f o a b s t r a c t

Article history: Our aim was to assess the potential of navigated transcranial magnetic stimulation (TMS)-evoked
Received 4 February 2008 electroencephalographic (EEG) responses in studying neuronal reactivity and cortical connectivity in
Received in revised form 25 March 2008 Alzheimer’s disease (AD) and in mild cognitive impairment (MCI). We studied 14 right-handed subjects:
Accepted 18 April 2008
five patients with AD, five patients with MCI and four healthy controls. Fifty TMS-pulses at an intensity of
110% of individually determined motor threshold were delivered to the hand area of primary motor cor-
Keywords:
tex (M1) with navigated brain stimulation (NBS). Spreading of primary NBS-evoked neuronal activity was
Alzheimer’s disease
monitored with a compatible 60-channel EEG, and analyzed in time, frequency and spatial-domains. We
Brain
Mild cognitive impairment
found significantly reduced TMS-evoked P30 (time-locked response 30 ms after the magnetic stimulation)
Navigated brain stimulation in the AD subjects. This reduction was seen in the temporo-parietal area ipsilateral to stimulation side
Primary motor cortex as well as in the contralateral fronto-central cortex corresponding to the sensorimotor network, which is
Evoked response anatomically interconnected with the stimulated M1. In addition, there was a significant decrease in the
Event-related response N100 amplitude in the MCI subjects when compared with the control subjects. Thus, the combination
of NBS and EEG revealed prominent changes in functional cortical connectivity and reactivity in the AD
subjects. This pilot study suggests that the method may provide a novel tool for examining the degree and
progression of dementia.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction
Electroencephalography (EEG) is a common tool for measur-
ing spontaneous and event-related electrical activity in the brain.
Transcranial magnetic stimulation (TMS) combined with compati-
ble EEG can be used individually and non-invasively to determine
functional connectivity within the brain by following the spread-
ing of electrical activity after locally applied stimuli (Ilmoniemi et
al., 1997; Kähkönen et al., 2001). Modern navigated brain stimu-
lation (NBS) systems also include navigation tools that locate the
focus of the TMS pulse in three-dimensional brain and allow precise
repeatability of location and stimulus intensity required averaging
stimulus-related EEG changes. Finally, high-resolution magnetic
resonance (MR) images suitable for 3D renderings of an individ-
∗ Corresponding author. Tel.: +358 44 7174118; fax: +358 17 173187.
E-mail address: petro.julkunen@kuh.fi (P. Julkunen).
ual brain provide the frame for evaluation of spatial reproducibility
and inter-individual comparisons.
Several distinguishable TMS-evoked responses in EEG, like P30,
N100 and P200, have been identified during last few years (Paus et
al., 2001; Tiitinen et al., 1999; Van Der Werf and Paus, 2006; Van
Der Werf et al., 2006). P30 has been suggested to involve pathways
between subcortical structures such as thalamic nuclei or basal
ganglia and cortex (Bonato et al., 2006). N100 has been shown to
relate partially to auditory-evoked response produced by the sound
during the discharge of the coil (Nikouline et al., 1999), and partially
by “real” cortical TMS-induced potentials (Komssi et al., 2004).
Alzheimer’s disease (AD) is the most common cause of demen-
tia in old age, but objective, early diagnostics has remained evasive.
Neuropathologically AD is characterised by neurofibrillary tangles
that first appear in the medial temporal lobe (MTL) structures
and progress in further stages through the limbic structures to
neocortical association areas (Braak and Braak, 1991). AD-related
pathology leads to degeneration of the large cortical pyramidal

0165-0270/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jneumeth.2008.04.021
 P. Julkunen et al. / Journal of Neuroscience Methods 172 (2008) 270–276 271

neurons (Hardy et al., 1986), presumably at the level of cortico- Table 1

Demographic and TMS-related characteristics (mean ± S.D.)
cortical projections. Correspondingly, a “disconnection syndrome”
has been proposed as a model of AD symptomatology (Morrison et Group Control MCI AD
al., 1986), and, indeed, neuroimaging studies have shown altered Subjects 4 5 5
functional connectivity in patients with AD (Grady et al., 2001). Age, years 77.8 ± 2.6 74.0 ± 8.1 73.2 ± 8.1
Mild cognitive impairment (MCI) has recently been proposed Female (%) 75.0 40.0 40.0
as prodromal AD (Petersen et al., 1995). Subjects with MCI have Education (years) 6.5 ± 1.0b 7.6 ± 3.0 9.6 ± 2.1
MMSE 27.0 ± 4.1b 25.4 ± 3.2b 22.0 ± 5.1a
an increased risk to develop AD with an annual conversion rate of
CDR total 0.0 ± 0.0† , ‡ , b 0.5 ± 0.0b 0.6 ± 0.2a
6–25% (Petersen et al., 2001). Recent imaging studies have found CDR sum of boxes 0.0 ± 0.0† , ‡ , a , b 1.9 ± 1.1 3.2 ± 2.5
atrophy of the MTL structures (Laakso et al., 1998; Pennanen et Stimuli (n) 52.5 ± 3.6 50.4 ± 0.5 49.3 ± 1.4
al., 2004) and reduced metabolism (De Santi et al., 2001) resem- MT intensity (%) 44.3 ± 15.7‡ 50.4 ± 12.4‡ , b 41.2 ± 4.6† , a
left hemisphere: 48.4 ± 10.8b 49.5 ± 13.3‡ , b 41.6 ± 4.0† , a
bling the findings of AD, whereas increased MTL activation has
right hemisphere: 40.2 ± 10.9† 47.7 ± 13.2 40.7 ± 5.6
been reported with functional magnetic resonance imaging (fMRI) Electric field strength (V/m) 82.6 ± 17.1‡ , b 80.6 ± 17.9‡ , b 61.3 ± 7.0† , a
in MCI subjects (Dickerson et al., 2004, 2005; Hämäläinen et al., left hemisphere: 85.5 ± 14.3b 82.4 ± 17.4b 63.2 ± 8.7a
2007). right hemisphere: 79.8 ± 21.3b 78.2 ± 20.5b 59.4 ± 5.1a
As altered functional connectivity may precede structural MEP amplitude (mV) 1.0 ± 0.6b 2.0 ± 2.6 2.9 ± 2.5
left hemisphere: 1.1 ± 0.6a , b 3.3 ± 3.6 2.5 ± 2.3
changes, an objective method for the investigation of early func-
right hemisphere: 1.0 ± 0.7b 1.0 ± 0.4b 3.3 ± 3.0a
tional changes would be useful in the diagnostics of MCI and
Motor-evoked potentials were measured from opponens pollicis muscle.
AD. Early identification of MCI would help aiming the current
Abbreviations: AD = Alzheimer’s disease, CDR = clinical dementia rating, MCI = mild
symptomatic AD-treatment to the appropriate subjects and in the cognitive impairment, MEP = motor-evoked potential induced by transcranial mag-
prospects of obtaining treatments that modify the course of AD, netic stimulation, MMSE = mini mental-state examination, MT = motor threshold.
it is even more important to accurately identify subjects likely to †
Significant difference (p < 0.05) as compared to the MCI group.
‡
convert to AD. Significant difference (p < 0.05) as compared to the AD group.
a
In this pilot study we investigated the potential of NBS-evoked Cohen’s d > 0.8 as compared to MCI group.
b
Cohen’s d > 0.8 as compared to AD group.
brain responses in the assessment of connectivity and/or reactiv-
ity changes related to AD and MCI. We hypothesized that impaired
functional connectivity and altered cortical reactivity of those dis- tory (Ransil and Schachter, 1994). No significant differences existed
eases could be revealed by focusing magnetic stimulation to a between the study groups in age or education (Table 1). Each sub-
functionally active and densely interconnected cortical area of the ject gave written informed consent, and the study was approved by
brain (primary motor cortex M1). If the results of this pilot study the local ethics committee.
indicate that the EEG-responses to M1 stimulation show local or
global differences in time- and/or frequency-domain in cognitively 2.2. Measurement setup
impaired subjects, the method may provide a new tool for investi-
gating early changes in MCI or AD. MR imaging was performed on a Siemens Magnetom Avanto
(Erlangen, Germany) 1.5T scanner. T1-weighted high-resolution 3D
2. Methods MR-images were used in the NBS. The stimulation setup consisted
of an NBS system (Nexstim Ltd., Helsinki, Finland) combined with
2.1. Study subjects a Magstim 200 magnetic stimulator (Magstim Ltd., Whitland, UK)
and a figure-of-eight monophasic TMS coil (Magstim Double 70 mm
We studied 14 voluntary right-handed subjects: five patients (PN6625)). During NBS, muscle activity was recorded and moni-
with AD, five patients with MCI and four healthy controls (Table 1). tored on-line by continuous electromyography (EMG) (ME 6000,
Both the controls and the MCI subjects were recruited from a lon- Mega Electronics Ltd., Kuopio, Finland), and EEG was recorded by
gitudinal population-based study (Hänninen et al., 2002; Tervo et a 60-channel TMS compatible EEG device (Nexstim Ltd., Helsinki,
al., 2004). Diagnosis of MCI required the following characteristics Finland). EMG was measured from pre-gelled disposable Ag/AgCl
(Petersen et al., 1995): (1) subjective memory impairment cor- electrodes attached to the skin overlying the thenar and hypothenar
roborated by an informant, (2) objective memory impairment as muscles. The NBS system delivered trigger pulses that synchronized
evidenced by a score of 0.5 in the clinical dementia rating (CDR) the TMS, EMG and EEG systems.
scale (Hughes et al., 1982) with at least 0.5 on the memory sub-
scale and a score of 1.5 S.D. below the average of a normative 2.3. TMS protocol
age-matched sample group in at least one memory test, (3) nor-
mal global cognitive function (MMSE ≥ 20) (Folstein et al., 1975), To locate the optimal stimulus site for the hypothenar mus-
(4) normal activities of daily living and (5) no dementia accord- cle, the hand muscle area around the anatomically defined “motor
ing to the NINCDS-ADRDA criteria (McKhann et al., 1984). All the knob” was mapped with high intensity stimulation. Stimulation
MCI subjects included in the study were classified as multidomain was started from the left or right hemisphere randomly for each
amnestic MCI (Gauthier et al., 2006). The five AD patients were subject. Stimulation intensities corresponding to MTs for both
recruited from the local neurological outpatient clinic, and they hemispheres in each subject (hand area on the primary motor cor-
underwent extensive diagnostic tests including neuropsychologi- tex, Fig. 1B) were determined at the optimal location with the
cal testing, laboratory sampling, MR imaging of the head as well as orientation of the coil such that the induced electric field was
a clinical and neurological examination. Diagnosis of AD was made perpendicular to the central sulcus. MTs were determined as was
according to the NINCDS-ADRDA criteria for probable AD (McKhann described in Säisänen et al. (2008). With the navigation software,
et al., 1984). None of the subjects had a history of neurological we computed the electric field strengths at the mapped optimal
or psychiatric disease other than AD or MCI. All the AD patients location in the cortex at MT intensity (Ruohonen and Ilmoniemi,
were using cholinesterase inhibitors, while no other subjects had 1999). This was done at the depth from the scalp where the grey and
medication affecting cognition at the time of NBS measurement. white matter threshold became visible. Then the stimulation was
Handedness was tested using the Edinburgh handedness inven- performed using a single-pulse mode with a stimulus intensity of
272 P. Julkunen et al. / Journal of Neuroscience Methods 172 (2008) 270–276

In addition, spectrograms were produced to show the

time–frequency characteristics of each group average. Analysis was
made for the CZ electrode as it was considered to capture the global
frequency characteristics in EEG. The spectrogram was made in a
window of 50 samples and with an overlap of 40 samples using Mat-
lab 6.5 (Mathworks Inc., Natick, MA, USA). The spectrograms were
also statistically compared between the groups in time–frequency
domain within the resolution yielded by the spectrogram func-
tion using Mann–Whitney U test. All these statistical analyses were
implemented with Matlab, and CURRY 4 was utilised for visualiza-
tion and current density computation (Compumedics, El Paso, TX,
USA).

2.5. Statistical analyses of demographic details

Fig. 1. (A) Electrode locations in the 60-channel EEG-measurement system and (B)
typical stimulation points presented with green crosses for one subject. The visu- The demographic data was statistically analyzed with SPSS soft-
alized cortical depth was 28 mm from the scalp. Central sulcus (CS) and precentral ware (version 11.5; SPSS Inc., Chicago, IL, USA). Due to non-normally
sulcus (pCS) have been visualized indicating the area of the primary motor cortex
in between.

110% × MT at the optimal site. Fifty stimuli were given repeatedly a

few seconds (>3 s) apart. The stimulation was performed separately
for each hemisphere.

2.4. EEG recording and analysis

The EEG was recorded with a 60-channel TMS-compatible

system (Virtanen et al., 1999) continuously throughout the exper-
iment. In the EEG system, a sample-and-hold circuit was applied
together with blocking of the amplifier input for 3 ms from the
stimulus to avoid amplifier saturation. An electrode cap was tightly
attached to the subjects’ head. All electrodes were referred to an
electrode placed on the right mastoid (Fig. 1A). The data were
recorded with a 1450-Hz sampling frequency and 16-bit preci-
sion. A trigger signal marking the exact stimulation moments was
recorded with the EEG. Electro-oculogram (EOG) was recorded
above and below the left eye.
In off-line analysis, the raw EEG data were filtered to a 0.1–25.0-
Hz frequency band. EEG data were divided to segments of 500 ms
including a 100 ms pre-stimulus baseline. Ocular correction was
applied to each segment (∼50 trials per subject) with Gratton
and Coles’ method utilising the EOG signal (Gratton et al., 1983).
Automated and visual artefact rejection for individual channels
was done before averaging the segments. A commercial software
package, Brain Vision Analyzer (Brain Products GmbH, Munich, Ger-
many) was used in the off-line analysis.
Group-averaged global-field-power (GFP, Lehmann and
Skrandies (1980)) curves were computed in order to reveal
the global time-dependent differences between the groups. The
average GFP for the groups was computed as a grand average
from each individual’s GFP computed with the data from all
measured (60 in maximum) EEG channels. To find the time periods
over which the differences between the groups were significant,
Mann–Whitney U test was point-wise applied to the GFPs.
For visualization of the scalp locations of the statistically signif-
icant differences between groups, a channel-wise Mann–Whitney
U test was applied to get a p-value for each electrode location.
Current density maps (based on minimum norm of each
group’s grand average data) were computed and visually compared
between the groups. Current densities were separately computed
for left- and right-side stimulation. A head model of three concen- Fig. 2. Global-field-power (GFP) waveforms (bold line) and statistical comparison
tric circles was used as a volume conductor model for the current for control and MCI group (A), for control and AD group (B) and for MCI and AD group
(C). The stimulation moment is presented with a dash line. The grey bars indicate
density map calculations. The current density mapping was done
the time ranges over which the waveforms differ statistically at a probability level
to aid visual comparison of the hypothesized cortical connectiv- of p < 0.05. The data from both hemispheres was pooled for the GFP analysis. The
ity/reactivity differences between the groups. GFPs were computed from as a grand average from each individual’s GFP.
 P. Julkunen et al. / Journal of Neuroscience Methods 172 (2008) 270–276 273

Fig. 3. Butterfly plots of TMS-evoked-potential curves for control (upper panel), MCI (middle panel) and AD (lower panel) group measured from both hemisphere stimulations.
Fifty single-pulse TMS were applied for each patient and hemisphere. Each trace represents the group average measured from individual electrodes.

distributed variables and small amount of subjects, non-parametric
Mann–Whitney test was used. To investigate the size of found
effects, we used Cohen’s d (d > 0.5 meaning medium effect and
d > 0.8 meaning large effect) (Rosnow and Rosenthal, 1996).
3. Results
The observed MTs were significantly (p < 0.05) lower in the AD
group than in the MCI group, and interestingly, the MCI subjects
showed the highest MTs of these three groups (Table 1). Instead, the
induced motor-evoked potential amplitudes were not significantly
different between the groups.
TMS evoked distinct P30, N100 and P200 potential peaks
(Figs. 2 and 3). There were significant (p < 0.05, d > 0.8) magnitude
differences in the GFPs between the AD group and controls between
30 and 90 ms as well as between 280 and 400 ms (Fig. 2). The
MCI group had a significantly (p < 0.05, d > 0.8) lower N100 GFP-
magnitude compared to the control group, and lower GFPs between
300 and 370 ms (d > 0.5). In addition, the GFP magnitude of P30
was significantly (p < 0.05, d > 0.2) decreased in the AD group as
compared to the MCI group (Fig. 2). These differences were visu-
ally apparent in the butterfly plots (Fig. 3). In particular, there
was focally reduced activity (not significant) in the 30 and 100 ms
latency range (P30 and N100, respectively) in the AD group and the
MCI group (Table 2). P30 was seen most clearly in the centroparietal
electrodes (CP1 and CP2) of the stimulation side. The N100 latency
was longer in the MCI group than in the other groups, but this dif-
ference was not statistically significant (Table 3). In addition, we

Table 2
Comparison of baseline-to-peak amplitudes of P30, N100 and P200 (mean ± S.D.)

Group P30 N100 P200

CP1 (␮V) left CP2 (␮V) right GA (␮V) combined CZ (␮V) left CZ (␮V) right GA (␮V) combined

Control 19.0 ± 12.3 15.6 ± 10.7 7.5 ± 3.5 −11.8 ± 5.3 −17.0 ± 9.4b 5.3 ± 4.4
MCI 17.0 ± 18.2 11.4 ± 14.2 7.6 ± 8.8 −9.0 ± 8.6 −11.3 ± 6.3a 4.9 ± 2.8
AD 11.8 ± 8.8a 7.0 ± 5.3a 3.8 ± 3.2a , b −4.6 ± 8.5a , b −10.6 ± 7.2a 5.0 ± 2.1

Potentials on the side of stimulation were measured from electrodes exhibiting highest amplitudes. P30 was most visible in electrodes close to stimulation, whereas N100
was most prominent at the vertex electrode (CZ ) and P200 was seen globally.
Differences between the groups were statistically insignificant.
GA = grand average of all electrodes.
a
Cohen’s d > 0.5 as compared to controls.
b
Cohen’s d > 0.5 as compared to MCI.
274 P. Julkunen et al. / Journal of Neuroscience Methods 172 (2008) 270–276

Table 3
Latencies for each group

Group P30 (ms) N100 (ms) P200 (ms)

Left Right Left Right Left Right

Control 31.0 ± 2.6 32.5 ± 1.7 94.3 ± 20.2 91.5 ± 15.9a 177.3 ± 11.0 182.3 ± 13.7
MCI 31.6 ± 1.8 32.3 ± 9.2 98.0 ± 9.0 102.0 ± 9.2b 176.5 ± 4.5 187.8 ± 16.2
AD 33.5 ± 1.7a , b 35.6 ± 1.5† , a , b 96.5 ± 8.1a 96.4 ± 10.1a 189.3 ± 10.4a , b 187.4 ± 8.7

Latencies were determined from the central CZ -electrode for stimulation of both hemispheres (mean ± S.D.). Stimulated hemispheres are indicated below the EEG-component
labels.
†
p < 0.05 according to paired samples Mann–Whitney test as compared to the control and the MCI group.
a
Cohen’s d > 0.5 as compared to MCI group.
b
Cohen’s d > 0.8 as compared to control group.

observed higher ipsilateral reactivity on the dominant (left) hemi- 4. Discussion

sphere for the TMS pulse applied to the same side in all the groups
(Table 2). TMS-evoked response at around 30–50 ms decreased signifi-
To evaluate the topographical differences in the evoked cantly over widespread brain areas in AD patients corresponding
potentials, a spatial significance map of TMS-evoked activation dif- to dysfunction of a large-scale sensorimotor network. This find-
ferences at 30 ms was computed between the control and AD group ing was consistent, whether measured globally using GFP, in the
(Fig. 4). Significant differences were seen on the ipsilateral parietal time–frequency domain using spectrogram or when visualized by
cortex, and on the contralateral frontotemporal areas. There were p-value mapping on the scalp or by cortical current density map-
no significant activation differences between the control and the ping.
MCI group at any location. The P200 showed no significant differ- Several studies have reported increased cortical excitability in
ences between any of the groups (Table 2, Fig. 2). AD patients and in increasing cognitive impairment (Alagona et al.,
Current densities were different between the groups. The dif-
ference was dependent on the stimulation side (Fig. 5). The current
density maxima were spatially located on the side of the stimula-
tion and temporally 30 ms after the stimulation. The difference in
the current densities was higher in the non-dominant than in the
dominant hemisphere.
The spectrograms (presenting spectral density of TMS-evoked
EEG in time and frequency domains in the vertex electrode) dif-
fered significantly between the AD and control group in two
time–frequency regions (Fig. 6): the AD group had a lower spec-
tral density at all major EEG-frequencies (<50 Hz) from 30 ms up to
100 ms and from 300 ms onwards. The MCI and control groups had
similar spectral densities.

Fig. 4. Topographic statistical p-value maps from Mann–Whitney U test between
control and AD groups at a latency of 30 ms. The green crosses represent the foci of
TMS-pulses. The lowest p-values for the left and right hemisphere stimulation were
0.06 and 0.04, respectively. No significant spatial differences were found between
the MCI and AD group or the controls and the MCI group.
Fig. 5. Current density maps for AD and control groups at a latency of 30 ms (P30
peak of the response). The scale range is normalized to the maximum current density
in the control group. The current maps were computed using CURRY4 with mini-
mum norm estimate and a head model of three concentric shells. The maps were
computed in the group level.
 P. Julkunen et al. / Journal of Neuroscience Methods 172 (2008) 270–276 275

Fig. 6. Spectrograms for control (A), MCI (B) and AD (C) groups in electrode CZ (Fig. 1A). Red indicates high and blue low spectral density. Statistical pixel-wise comparison
between control and MCI (D), control and AD (E), and MCI and AD (F) is presented. For the analyses of spectrograms, off-line filtering was not applied. In AD group, from
30–90 ms as well as after 300 ms latency spectral density was significantly reduced at all frequencies indicating lower activity reflecting reduced connectivity. In the right
panel (D–F) estimated regions of interest for significant differences are indicated by blue boxes and the stimulation moment by a dashed red line.

2001; de Carvalho et al., 1997; Di Lazzaro et al., 2004, 2002; Ferreri
et al., 2003; Nardone et al., 2006; Pennisi et al., 2002). Similarly, we
found the lowest MT in the AD group (Table 1).
All the basic components in the TMS-evoked EEG response were
visible in AD, MCI, and normal groups, but the early responses pre-
sumably reflecting cortical reactivity and functional connectivity
were decreased in AD patients. The TMS-evoked P30 peak (Paus et
al., 2001; Tiitinen et al., 1999) was lower in the AD group than in
the control and MCI groups (Fig. 2, Table 2) in the present study.
In addition to or instead of diminished reactivity and/or connectiv-
ity between brain regions, the decreased responses may also mean
that the EEG activity in the AD group was less synchronized than
in the controls or MCI, a finding which is in line with a previous
report showing that the EEG synchronization decreases in relation
to cognitive impairment (Koenig et al., 2005).
P30 and P200 amplitudes of the MCI group were about halfway
between the values of the AD and control group which is in line with
the current concept of MCI being a transition state from healthy
aging to AD (Petersen et al., 1995). However, the MCI group showed
slightly increased activity in the P30 GFP magnitude. In recent
studies, increased fMRI activation has been found in MCI subjects
(Dickerson et al., 2004, 2005; Hämäläinen et al., 2007) and corre-
spondingly, another study suggested that the cholinergic activity in
MCI subjects may actually be increased (DeKosky et al., 2002). Thus,
the MCI subjects may be in a “compensatory” hyperactive cholin-
ergic state leading to exaggerated neuronal responses to extrinsic
stimuli. The MCI group also had the highest variation in TMS-locked
EEG activation (Table 2), which may reflect the intrinsic heterogene-
ity of the MCI (Gauthier et al., 2006). The small number of subjects
in this study as well as the heterogeneity among MCI subjects war-
rants future research with larger sample sizes.
The magnitude of TMS-evoked EEG responses was considered
to reflect predominantly the synchronized activation of underlying
neuronal population activity and thus the functional connectivity of
the neuronal pathways originating from M1. Highest observed peak
amplitudes (P30, N100 and P200), that have also been detected in
several other studies (Bonato et al., 2006; Nikouline et al., 1999;
Paus et al., 2001; Tiitinen et al., 1999) were presumed to provide a
measure of functional connectivity from the site of magnetic stim-
ulation to the sources of these evoked responses. Alterations in
connectivity were clearest in the spatial p-value map between the
AD and control group (Fig. 4).
The synchronization of EEG was considered to reflect the con-
nectivity of the neuronal pathways, whereas the magnitude of GFP
was suggested to reflect subjects’ intrinsic reactivity to TMS. The AD
group had a lower spectral density of EEG at CZ at 30 ms post-stimuli
than in the other groups (Fig. 6). This is in good agreement with pre-
vious studies showing reduced, spontaneously synchronized alpha
and beta activity in AD patients (Huang et al., 2000).
The TMS-pulse was found to produce a large artefact. A similar
effect has been observed in other studies (Julkunen et al., 2008;
Paus et al., 2001; Raij et al., 2008). Therefore, the first 20 ms after
the TMS pulse were excluded from the statistical analyses. Click-
induced auditory responses observed by Nikouline et al. (1999)
were minimized by using earplugs. However, we found clear differ-
ences in N100 latencies between the groups. Previously, defects in
cholinergic system have been related to prolonged auditory N100,
which may partly explain the delay in N100 observed in AD group
(Pekkonen et al., 1999, 2005).
Despite the small group sizes in the present pilot study, we found
statistically significant changes in cortical connectivity and reac-
tivity in the AD patients. These changes should be studied in the
future with a larger population in both AD and MCI. However, in the
present study, the proof of concept for the potential of NBS–EEG in
AD research was reached.
Conflicts of interest
Jari Karhu works part-time as professor in the Faculty of
Medicine at University of Kuopio and part-time as Chief Medical
276 P. Julkunen et al. / Journal of Neuroscience Methods 172 (2008) 270–276
Officer in Nexstim Ltd., manufacturer of navigated brain stimula-
tion instruments. The recordings and analysis of the present study
were performed solely in the premises of the Kuopio University
Hospital by the other authors.
Acknowledgements
This study was supported by the National Technological Agency
of Finland (TEKES) Helsinki, Finland and an EVO (5772739) grant
from Kuopio University Hospital.
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