Eyes Wide Shut: Amygdala Mediates Eyes-Closed Effect
on Emotional Experience with Music
Yulia Lerner1,2, David Papo2, Andrey Zhdanov2, Libi Belozersky2, Talma Hendler2,3*
1New York University, Center for Neural Science, New York, New York, United States of America, 2Functional Brain Imaging Unit, Tel Aviv Sourasky Medical Center, Tel
Aviv, Israel, 3Tel Aviv University, Tel Aviv, Israel
The perceived emotional value of stimuli and, as a consequence the subjective emotional experience with them, can be
affected by context-dependent styles of processing. Therefore, the investigation of the neural correlates of emotional
experience requires accounting for such a variable, a matter of an experimental challenge. Closing the eyes affects the style
of attending to auditory stimuli by modifying the perceptual relationship with the environment without changing the
stimulus itself. In the current study, we used fMRI to characterize the neural mediators of such modification on the
experience of emotionality in music. We assumed that closed eyes position will reveal interplay between different levels of
neural processing of emotions. More specifically, we focused on the amygdala as a central node of the limbic system and on
its co-activation with the Locus Ceruleus (LC) and Ventral Prefrontal Cortex (VPFC); regions involved in processing of,
respectively, ‘low’, visceral-, and ‘high’, cognitive-related, values of emotional stimuli. Fifteen healthy subjects listened to
negative and neutral music excerpts with eyes closed or open. As expected, behavioral results showed that closing the eyes
while listening to emotional music resulted in enhanced rating of emotionality, specifically of negative music. In
correspondence, fMRI results showed greater activation in the amygdala when subjects listened to the emotional music
with eyes closed relative to eyes open. More so, by using voxel-based correlation and a dynamic causal model analyses we
demonstrated that increased amygdala activation to negative music with eyes closed led to increased activations in the LC
and VPFC. This finding supports a system-based model of perceived emotionality in which the amygdala has a central role
in mediating the effect of context-based processing style by recruiting neural operations involved in both visceral (i.e. ‘low’)
and cognitive (i.e. ‘high’) related processes of emotions.
Citation: Lerner Y, Papo D, Zhdanov A, Belozersky L, Hendler T (2009) Eyes Wide Shut: Amygdala Mediates Eyes-Closed Effect on Emotional Experience with
Music. PLoS ONE 4(7): e6230. doi:10.1371/journal.pone.0006230
Editor: Hans P. Op de Beeck, University of Leuven, Belgium
Received December 28, 2008; Accepted June 10, 2009; Published July 15, 2009
Copyright: ? 2009 Lerner et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by the US-Israel Bi-national Science Foundation (TH). The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
The perceived emotional intensity of stimuli can determine its
assigned adaptive value by the individual. It has been acknowl-
edged that perceived emotionality of stimuli is highly dependent
on style of processing (e.g. focus of attention), perceptual context
(e.g. the relatedness to environment) and task relevance, which
together form the individual’s mental set . Exploring the neural
marking of a change in style of processing may reveal the
underlying mechanism of the phenomenal aspect of emotional
experience. However, this calls for experimental probes of shifts in
style of processing while keeping unchanged the physical
properties of the stimulus used as a carrier for emotions. Closing
one’s eyes while listening to sound may serve such aim as it evokes
shifts in style of processing by modifying focus of attention, while
keeping targeted stimuli the same. The main outcome of such a
shift could enhance the perceived intensity of emotional stimulus,
making positive attributes more positive and negative ones more
negative. For instance, the positive feeling of relaxation is
facilitated by closing the eyes; on the other hand, when closing
the eyes in the presence of negative-valued external stimuli,
subjects tend to increase their preparedness to a possible threat.
Brain wise, the closed eyes position is well known by its unique
electrophysiological signature of increased alpha rhythm . In a
recent resting state study, fMRI and EEG signals were recorded
simultaneously while participants intermittently were instructed to
close or open their eyes. Using the combined measurements, two
alpha rhythms were defined tempo-spatially with relation to the
closed eyes position; one on-going and spontaneous distributed in
the midline brain regions including the prefrontal cortex (PFC),
cingulate cortex and thalamus, and the other stimulated by shift in
eyes position from open to closed distributed in the fronto-
temporal cortical regions. These data point to the possibility that
closing the eyes indeed characterizes a specific brain state that can
be affected by the individual’s mental set . Accordingly, the
current study presumes that eyes closed position represents a well
defined mental set by which perceived emotionality can be
modulated, thus probing its neural respect.
Changes in perceived emotionality associated with a given
stimulus can be related to modification in the processing of
visceral- or cognitive emotional cues, through activation of
vigilance- or appraisal-based mechanisms, respectively. The
importance of visceral-related cues in the primacy of emotional
processing was originally suggested by James  and more
recently updated as the ‘somatic marker hypothesis’ by Damasio
. On the other hand, a critical involvement of cognitive schema
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of appraisal in the establishment of emotional experience has been
originally put forth by Schachter and Singer  and more recently
emphasized from a clinical perspective by Beck and Clark . It
seems though, that people vary tremendously in their tendencies to
rely on either of these processes when encountering an emotional
stimulus in the environment. While some individuals described as
‘aware of their visceral cues’, tend to be more emotionally
expressive and to experience emotions as more intense [8,9],
others tend to be over-occupied with internally generated self-
focused negative ruminations and suffer from depressive symptoms
. This inter-individual variability calls for a certain interplay
between visceral and cognitive based neural operations of
emotion, as nicely suggested recently by the ‘conceptual act
model’ . This model posits that our aware emotional
experience emerges out of an ongoing flux of a ‘core affective
state’ (i.e. the experience of a pleasant/unpleasant feeling with a
varying amount of arousal) as conceptualization (i.e. appraisal,
reflection) of this state takes place. Furthermore, those two
processing stages assumed to be mediated by different brain
networks, involving ‘lower’ (i.e. independent of directed attention
and awareness) and ‘higher’ (i.e. depends on directed attention and
awareness) levels of brain operations, respectively. Interactions
within and between these levels may play a key role in the
establishment of the unique subjective emotional experience out of
an ongoing background feeling state.
The neural apparatus mediating the processing of affective
attributes of stimuli (i.e. perceived emotionality) has been
extensively studied in humans, although the modulating effect
of the perceived intensity has hardly been examined. The
amygdala together with its dense connections with both
prefrontal cortex and the brainstem has been put forth as a core
region in this respect . Projections of the amygdala to
brainstem areas such as the LC were proposed as instrumental in
the detection of and orienting to visceral signals of emotions .
Recent imaging studies further support the relatively automatic
nature of emotional processing via this path, by showing a closer
relation between the amygdala and the LC activation during
unaware than aware processing of negative facial expressions
The amygdala’s dense connections with the VPFC have been
repeatedly demonstrated in animals [15–17]. More recently,
imaging studies demonstrated a correlation between activations
in the amygdala and the PFC including its ventral aspect in
relation to appraisal of negative emotional stimuli [18,19].
Importantly, neuroanatomical and physiological studies also show
that the PFC is heavily projecting to the LC. For example, the
monkey LC has been shown to receive strong direct projections
from various medial prefrontal regions thought to evaluate and
monitor task-related contexts . Accordingly, these authors
proposed recently that LC might be responsive to ongoing
evaluations of task-related appraisal provided by inputs from the
PFC. Subsequently, LC modulates cortical sites as a function of
the appraisal processes, thus selectively adjusts subjects’ mental set
towards task- or stimulus-specific processes. Yet, the mode of
interplay between ‘low’ and ‘high’ levels of neural operations for
determining the perceived emotional value is poorly understood.
In the current study we investigated whether closing the eyes
while keeping stimuli and task unchanged, modifies the perceived
intensity of the emotional value conveyed by various musical
excerpts. Music has commonly been used as an emotional stimulus
due to its highly affective, though abstract, value in humans .
Interestingly, most imaging studies failed to demonstrate amygdala
activation to emotion in music [22,23], but see [24,25]. In a recent
study in our lab we found that adding emotional music to a short
neutral video clip resulted in increased emotional value of the
movie and greater activation in the amygdala than with the
presentation of the neutral movie or emotional music alone . It
is not clear though if this combined presentation effect in the
amygdala was due to increased semantic cues or greater
complexity of the emotional stimuli (i.e. multi-modality). Here,
we are able to manipulate the perceived emotionality via change in
the mental set (i.e. eyes position) while keeping physical aspects of
the emotional stimuli unchanged (i.e. music). This way we could
test separately the effects of perceived emotional value (i.e.
negative vs. neutral), processing context (eyes open vs. eyes
closed), and their interaction (change in perceived intensity with
regard to eyes position). For that, we performed behavioral and
fMRI experiments using short music clips of negative and neutral
emotional values (Stimuli S1, S2, S3, S4, S5, S6, S7, S8) presented
in three context conditions (see Figure 1A): when subjects’ eyes
were (i) closed, (ii) open while viewing a black screen, (iii) open
while viewing a scrambled video clip. In a separate fMRI
experiment we examined the effect of closing and opening the
eyes in complete darkness (see Materials and Methods). We
assumed that the eyes closed position would enhance perceived
emotional intensity of the musical excerpts, more so for the
negative pieces, and that this would be associated with increased
activation in the amygdala, as well as with greater functional
connectivity between the amygdala, the LC and VPFC;
representatives of the ‘low-visceral’ and ‘high-cognitive’ neural
operations of the human emotional experience.
Behaviorally the experiment was set to answer to what extent
closing the eyes affects the experience of emotion in music.
Figure 1B–D show that there was a significant difference in
valence rating between closed and open eyes states only for the
negative musical clips, with a higher rating for the closed eyes state
(one-tail t-test: ‘negative closed’ vs. ‘negative open’: t(11)=22.1,
p,0.05; ‘negative closed’ vs.
t(11)=23.2, p,0.01). Moreover, there was a similar difference
for closed vs. open eyes for the rating of arousing value of negative
music (one-tail t-test: ‘negative closed’ vs. ‘negative open’ and
‘negative closed’ vs. ‘negative scrambled-movie’: t(11)=22.04,
p,0.05). In contrast, no such difference was obtained for the
rating of the level of abstraction in the stimuli.
fMRI: Activation Distribution and Magnitude
Analyzing the imaging data we first performed a whole brain
analysis to delineate the eyes-state related networks. The results of
this analysis are summarized in Table 1. Closed eyes vs. open eyes
across music conditions revealed activation in the VPFC, superior
temporal cortex as well as in the limbic areas such as the
amygdala, anterior hippocampus, temporal pole and cingulate
cortex. In contrast, open eyes vs. closed eyes showed as expected
preferential activation in perceptual regions in the occipital,
parietal and lateral temporal regions as well as in the dorso-lateral
PFC (DLPFC). This analysis highlighted an interesting dissociation
in PFC activation in regard to mental set induced by eyes position.
Figure 2 shows the striking functional segregation in the PFC
between eyes positions on inflated group activation map (N=12,
p,0.05, random effect); closed eyes were associated with
activation of VPFC areas (BA 47 and BA 11), while open eyes
evoked activity in the DLPFC areas (BA 46/9 and BA 10).
Talairach coordinates and voxel extension are presented in Table
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Since this study was focused on the effect of shifting eyes
position on emotional experience we examined the difference
between closed and open eyes in relation to each music type
separately (i.e. neutral and negative). Figure 3 shows the results
from whole brain analysis on two views of group maps (N=12,
p,0.05, random effect). It can be seen that during negative music
but not during neutral music closed eyes evoked greater activation
than open eyes in the amygdala/anterior hippocampus complex
and anterior temporal poles. This pattern was highly consistent
across all subjects as shown by individual’s activation maps (Figure
Figure 1. Experimental design and behavioral results. (A) A segment from the time axis of the experiment. (B)–(D) Results of psychophysical
tests conducted outside the magnet on the same subjects who participated in the fMRI study and using the same stimuli. Evaluation of the emotional
valence (B), arousal (C) and abstraction (D) levels of the clips are presented. Stimuli types (x-axis) are identified in the apertures above the graphs.
Note a significant difference only for negative clips presented with eyes closed. Y-axes represent emotional dimensions. *, p,0.05; **, p,0.01. Error
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S1). In contrast to limbic areas, increased activation in the VPFC
for closed eyes relative to open eyes was seen for both negative and
neutral music types.
It is interesting to note that the VPFC foci showed a slight
trend toward greater selectivity to emotion when subjects were
instructed to judge the emotionality of musical clips (% signal
change, negative, closed: 0.6260.26 with task vs. 0.5260.19
without task; open: 0.1660.31 vs. 0.0960.03; movie: 0.0460.15
vs. 0.0260.01; neutral, closed: 0.4360.14 vs. 0.4260.16; open:
20.0260.08 vs. 20.0460.1; movie: 20.160.1 vs. 20.1160.08).
The amygdala was unaffected by this instruction (% signal
change, negative, closed: 0.6960.35 with task vs.0.760.2
without task; open: 20.1860.13 vs. 20.1860.04; movie:
20.3260.14 vs. 20.3160.16; neutral, closed: 0.260.2 vs. 0.2
0.1; open: 20.1960.07 vs. 20.260.16; movie: 20.0960.2
To examine the magnitude of limbic areas’ recruitment by the
closed eyes position we performed a region of interest (ROI)
analysis on our a-priori assumed core limbic area – the amygdala,
and on spatially associated area – the anterior hippocampus (see
Table S2 for Talairach coordinates). Figure 4A depicts the
sampled activation level in the amygdala (based on internal
localizer approach, see Materials and Methods) averaged across
twelve subjects. An activation profile for the hippocampus was
highly similar to that observed for the amygdala. As no main effect
for hemisphere was found, we collapsed the data from the left and
right hemispheres. A one-way ANOVA was performed within
each music type (neutral, negative) with the three eyes position
conditions (eyes closed, eyes open and scrambled movie) for the
(F(1,11)=22.9, p,0.001). Further, a post-hoc analysis (Fisher’s
PLSD) revealed a significant difference between the closed eyes
position and the other two open eyes conditions for the negative
music type both in the amygdala (p,0.001 in tests: ‘open vs.
closed’ and ‘scrambled-movie vs. closed’) and the anterior
hippocampus (p,0.001 in tests: ‘open vs. closed’ and ‘scram-
bled-movie vs. closed’). For the neutral music a significant
difference between eye states was achieved in the anterior
hippocampus (p,0.001 in tests: ‘open vs. closed’ and ‘scram-
bled-movie vs. closed’) but not in the amygdala. Thus, as shown by
the post-hoc analysis of the ROI data a tendency for a stronger
interaction between eyes position and music type was present in
the amygdala than in the hippocampus. To quantify this effect
within the amygdala we calculated an index of eyes-state: (close –
open)/(close+open) separately for negative and neutral music. The
analysis revealed significant difference between the ratios for
negative and neutral music (t(11)=2.7, p,0.05, paired t-test)
(Figure 4B). Together, as expected the whole brain and ROI
analyses demonstrated a closed eyes effect in core limbic regions
such as the amygdala and anterior hippocampus.
Figure 2. Differences in brain activation with eyes closed, eyes open and activation profiles of ROI. Functional averaged activation maps
(N=12, p,0.05, random effect) show the cortical activity evoked by musical clips (negative and neutral) presented with eyes closed (blue) and open
(green). The maps are superimposed on the left (LH) and right (RH) unfolded hemispheres shown in the lateral view. Quantitative analysis of the
activation levels is shown for open (green patches on the maps) and closed (blue patches on the maps) eyes. While the BA 47 (‘blue-framed’ bars,
blue circle on the map) exhibited highly preferential activation for eyes closed, the BA 46/9 (‘green-framed’ bars, green circle on the map)
demonstrated highly preferential activation for eyes open. STS – superior temporal sulcus, IPS – intraparietal sulcus, PCS – post-central sulcus, LS –
lateral sulcus, IFS – inferior frontal sulcus. Error bars, SEM. *, p,0.05.
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We then asked if the observed emotional selectivity of the closed
eyes effect (i.e. ‘closed vs. open’ for negative music) in the
amygdala corresponds to change in the emotional experience of
these music clips. We investigated this possibility by correlating
between the closed and open eyes differences in behavioral and
brain measures in the amygdala separately for the negative and
neutral music clips. Significant correlation was found only for the
amygdala between difference in measure of activation (negative
closed – negative open) and difference in the arousal intensity for
negative (R2=0.5405, p,0.05, Figure 4C) but not neutral music
clips (p=0.3). This finding raises the possibility that the increased
activation for negative music clips presented with eyes closed in the
amygdala being related to a modified emotional experience as
expressed by reports of increased arousing character of the music.
Additionally, an expected effect of context was observed.
Significant correlation was found in the amygdala between
difference in brain measures (negative closed – neutral closed)
and difference in the arousal intensity for eyes closed (R2=0.5888,
p,0.05, Figure 4D) but not for eyes open state (p=0.5).
In order to estimate the regional specificity of emotional
modulation on eyes closed effect we performed a ROI analysis in
three main foci of PFC activation obtained from eyes-state effect
contrast: BA 47, BA 11 and BA 46/9 (see Figure 2 for distribution
and Table S2 for Talairach coordinates). Again, no differences
between hemispheres were found, thus the data were collapsed
from both hemispheres. In the BA 47 a one-way ANOVA
performed within each emotion revealed an effect (F(1,11)=3.6,
p,0.05) of eyes-state for both types of emotions - greater activation
for closed eyes than open eyes for the negative as well as for the
neutral emotions. Similarly, in the BA 11 the difference between
closed and open eyes states was significant (p,0.01) for the
negative and neutral emotions. In contrast, the BA 46/9
demonstrated a significantly (F(1,11)=2.7, p,0.05) preferential
activation for open eyes compared to closed eyes within negative
and neutral emotion types. These findings demonstrated that
within the PFC there is evidence for differential processing of the
same music depending on the eyes state. Note that preference for
emotion type was also opposite in these regions: while the BA 47
and BA 11 showed a preference for negative clips with eyes closed,
the BA 46/9 demonstrated a preferential trend for neutral ones
with eyes open (Figure 2).
fMRI: Functional Connectivity
Having revealed an intriguing activity modification in the PFC
and the amygdala depending on the mental set induced by eyes
positions, we were interested in examining to what extent these
effects can be explained by co-activation of these regions with other
brain areas. To that end, we performed functional connectivity
analysis with ‘seed’ time courses of preferential activation derived
from the PFC ROIs (BA 47 for eyes closed, BA 46/9 for eyes open,
Figure 5A) or the amygdala ROI (eyes closed with negative music).
We then used these time courses as the General Linear Model
(GLM) predictors to compute a voxel-by-voxel fit (see Materials and
Table 1. Whole-brain activation analysis.
Talairach coordinates (mm)
open vs. close (random effect)
Early Visual Areas17, 18
Precuneus 7, 19
Intraparietal Sulcus 39, 7
Superior Parietal Lobule7 26
Superior Temporal Sulcus22
Middle Frontal Sulcus9, 46
2398 32 4237 25p,0.008
Middle Frontal Gyrus 9,10
235469 4046 14p,0.001
Superior Frontal Gyrus 10
29 62 31 266119p,0.0005
close vs. open (random effect)
Superior Temporal Sulcus22
Orbital Sulcus 11
26 17 48
21 32 32
Cingulate Gyrus 247
Anterior Superior Temporal Gyrus38
Areas that exhibited the significant levels of activation during stimulation by the eyes closed and the eyes open states.
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Methods for details), separately for each region. Fit evaluation
revealed an intriguing effect: activation in the BA 47 was correlated
with activation in the amygdala/anterior hippocampus complex
and in the superior and middle temporal sulci and gyri (Figure 5B,
p,0.001). The BA 11 also was tested for the eyes closed; the pattern
of its connectivity was highly similar to that observed for the BA 47
(p,0.05, not shown). When the time course from the BA 46/9 was
used as a predictor, activation was correlated mainly with
distributed activation in the visual-related areas (Figure 5C,
p,0.001). In order to quantify these mapping findings we
conducted a direct statistical comparison between these two
connectivity analyses. Figure 5Ddepicts that the correlated activity
of the VPFC and amygdala was significantly stronger than the
correlated activity of the DLPFC and amygdala, corresponding to
mental sets of eyes closed and open, respectively (z score=7.99,
emotion in music we repeated the analysis with contrast time course
obtained for each emotion separately, i.e. ‘seed’ time courses were
defined once for the negative music and once for the neutral music
(tests: ‘closed negative . open negative’, ‘open negative . closed
negative’, ‘closed neutral . open neutral’, ‘open neutral . close
neutral’). Statistical analyses of connectivity differences between
eyes closed and eyes open states revealed significant differences for
negative music (z score=7.72, p,0.001) as well as for neutral music
(z score=3.4, p,0.001) for both VPFC and DLPFC. Moreover, a
direct statistical comparison between the co-activation in the
negative and neutral conditions was performed for the VPFC and
DLPFC (Figures 5E–F). Although for both areas the co-activation
Figure 3. The effect of eyes closed on the amygdala. Average activation patterns (N=12, p,0.05, random effect GLM analysis) revealed by the
contrast ‘eyes closed . eyes open’ and superimposed on the coronal and transversal views. Significant activation was found in the amygdala/anterior
hippocampus complex (orange arrows) for the negative clips (left panel) but not for the neutral ones (right panel). The color scale indicates
significance level. L – left hemisphere, R – right hemisphere, COR – coronal, TRA – transversal.
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Figure 4. Sensitivity to emotional clips as reflected in activation profiles. (A) Average activation levels obtained in the amygdala. Stimulus
type (x-axis) is indicated in the apertures above the graphs. The y-axis denotes an fMRI percent signal change relative to blanks. Asterisks denote a
significant difference between eye states; p,0.001. Error bars, SEM. (B) ‘Index of eye state’ expressed by formula (close – open)/(close+open) indicates
the ratios for negative and neutral stimuli. Asterisk denotes a significant difference between emotions; p,0.05. Error bars, SEM. (C) Relationship of the
fMRI data and behavioral performance. Significant correlation (p,0.05) was found only for the amygdala between difference in measure of activation
(negative closed – negative open, %SC – % signal change) and difference in the arousal intensity for negative clips. (D) Significant correlation
(p,0.05) was found in the amygdala between difference in brain measures (negative closed – neutral closed, %SC – % signal change) and difference
in the arousal intensity for eyes closed state.
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Figure 5. Correlation analysis for the PFC ROIs. (A) ‘Seed’ time courses defined separately for eyes open and closed are shown on the parts of
unfolded hemispheres. ‘Seed’ time courses were used to compute a voxel-by-voxel fit for the (B) BA 47 and for the (C) BA 46/9. Note a strong
connection of the amygdala to the BA 47. The color scale indicates significance level. R – right hemisphere, L – left hemisphere. (D) Correlated activity
of the VPFC (BA 47) and amygdala in comparison to the correlated activity of the DLPFC (BA 46/9) and amygdala, corresponding to mental sets of
eyes closed and open, respectively. Note a significant difference between correlation coefficients. (E–F) Co-activation analyses respectively to the
emotional context in music. A statistical comparison between the co-activation in the negative and neutral conditions was performed for the BA47 (E)
and BA 46/9 (F).
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was significantly stronger for negative music than neutral music, the
correlation derived from the BA 47 was much stronger (z
score=5.18, p,0.001 vs., z score=2.44, p,0.01). The computed
correlation coefficients for each subject and different conditions are
provided in Table S3. These data demonstrate the consistency in
the effect among subjects.
The correlation analysis with ‘seed’ time course from the
amygdala during eyes closed condition revealed an additional
striking network of co-activation that was most prominent
(p,0.001) in the bilateral pontine brainstem nuclei including the
LC, ventral striatum areas including nucleus accumbens (NAcc)
and anterior temporal gyrus region (Figure 6A, C, D). Again, we
checked this co-activation respectively to the emotional context,
using the amygdala once as a negative ‘seed’ obtained in the test
‘closed negative vs. open negative’ and once as a neutral ‘seed’
obtained in the test ‘closed neutral vs. open neutral’. As expected,
statistical analyses revealed that the correlated activity between the
amygdala and LC during eyes closed is much stronger in the
negative emotional condition than in the neutral condition (z
score=39.73, p,0.001, Figure 6B).
Finally, to further quantify the connections between the PFC,
amygdala and LC we applied the dynamic causal modeling
(DCM) tool using SPM2. The DCM was aimed to examine two
issues that were left open following the effective connectivity
analysis. (i) Does the amygdala lead the enhanced inter-regional
connectivity during the mental set of eyes closed? (ii) Is this
enhanced connectivity modulated by emotionality in music and/
or mental set of eyes closed?
The results for the first issue are presented on a brain model
depicting the direction and strength of intrinsic connections by
probability and intensity measures. Figure 7Ademonstrates that
during the mental set of eyes closed and negative music the
connectivity from the amygdala toward the PFC was more
probable and stronger (95%, 0.51 Hz respectively) than from the
PFC to the amygdala (72%, 0.18 Hz, respectively). Similarly, the
connectivity from the amygdala to LC was more probable and
stronger (86%, 0.39 Hz, respectively) than from the LC toward the
amygdala (67%, 0.09 Hz, respectively). Paired t-tests confirmed
the significance of these differences in connectivity between
directions (p,0.001, for the amygdala-PFC vs. PFC-amygdala
and the amygdala-LC vs. LC-amygdala). For the second issue we
performed a two-ways ANOVA on measures of probability and
strength for stimulus entrance (i.e. direct effect) to the amygdala,
with regard to emotionality of music (i.e. negative, neutral) and
mental set (i.e. closed, open). Figure 7Bshows that the eyes closed
state led to high probability and strength of music entrance to the
amygdala (F(1,13)=16.45, p=0.001), more so with negative than
neutral valence (interaction values F(1,13)=4.58, p=0.05). Post-
hoc analysis (Fisher’s PLSD) revealed a significant difference
between the eyes state under both negative (p,0.001) and neutral
music (p,0.01) but there was a significant difference between the
music types only under the closed eyes state (p,0.05). Finally, we
examined whether the music itself modulates the inter-regional
connections. This analysis revealed that music significantly
modulates the connections of the amygdala to PFC (eyes closed:
negative 20.589, neutral 20.622, eyes open: negative 20.613,
neutral 20.537) and amygdala to LC (eyes closed: negative
20.577, neutral 20.605, eyes open: negative 20.559 neutral
20.545), albeit disregarding mental set or emotionality. All these
effects were significantly greater than chance (p,0.001) but no
significant difference was found between them. In other words,
there was no evidence for selectivity in the modulation exerted by
music on the connections between the amygdala toward the PFC
and the LC.
fMRI: Control Experiment
Closed eyes state is also associated with shutdown of visual
input. This raised the possibility that the observed effect of eyes
closed was driven by the corresponding effect of darkness. Our
original experiment involved a condition of black screen during
the eyes open state. Although this condition by itself did not
produce the closed eyes effect seen in respect to the gray screen
(see Figures 2, 3), it could still be claimed that it is a matter of
magnitude and that black screen is not a good enough comparison
for visual input shutdown. We tested this possibility in a control
study where musical clips were presented in a complete darkness
while subjects were asked to either open or close their eyes (see
Materials and Methods). The whole brain analysis of this
experiment revealed a similar closed eyes effect as our original
experiment with strong preferential activation in the amygdala/
anterior hippocampus complex, anterior temporal pole and VPFC
for eyes closed (Figure S2 A). By sampling signals in the amygdala
and the anterior hippocampus we could estimate a quantitative
measure of this effect (Figure S2 B, an internal localizer approach
was used, see Materials and Methods). In complete darkness, one
would expect to have more activation in the amygdala in response
to the negative music as compared the neutral music, indepen-
dently of the eyes state. Indeed, relative to our original experiment,
overall higher activation to music with eyes open was found
(compare Figure S2 B and Figure 4A). Moreover, activation to
neutral stimuli with eyes closed notably increased as compared to
the original experiment. However, a central effect of significant
prevalence of the amygdala and hippocampus activation with eyes
closed over eyes open state remained for both negative (amygdala:
(t(5)=1.8, p,0.05; hippocampus: t(5)=2.2, p,0.05) and neutral
stimuli (amygdala: t(5)=3.8, p,0.05; hippocampus: t(5)=2.3,
p,0.05). These results confirm that the overall closed eyes effect
cannot be explained solely by the corresponding shutdown of
Our behavioral results demonstrated that closing the eyes
enhances the unpleasant and arousing character of passively heard
music excerpts. Although music did not vary, the experience of its
emotionality differed following a mental set shift induced by
moving from eyes open to eyes closed state. The neuroimaging
results showed increases in activation with eyes closed in the
amygdala, anterior hippocampus, temporal pole, and VPFC foci.
Remarkably, only in the amygdala was this effect selective to music
emotionality showing greater response to negative music with eyes
closed than open. It can, therefore, be concluded that context-
specific styles of attending can modify amygdala’s activation in
response to emotional music. Furthermore, correlation analysis
with amygdala’s activation when listening to negative music with
eyes closed showed co-activation with the LC and the VPFC. This
buttresses our assumption that, even in the absence of stimulus
changes, a shift in mental set may affect emotional perception by
parallel recruitment of distributed neural operations related to
‘low-visceral’ as well as ‘high-appraisal’ related processing,
respectively. Here, we discuss how such amygdala driven inter-
level neural recruitment might affect the subjective emotional
The Effect of Eyes Closed on Amygdala Activation
Although behaviorally emotional music excerpts were rated as
more negative and arousing than neutral sound excerpts in both
open and closed eyes states, selectivity to emotionality in the
amygdala was shown only in the latter (Figures 3–4). The low
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sensitivity of the amygdala to the emotional value associated with
music presented with eyes open is in line with prior PET and
fMRI studies in humans [22,23,27]. Though opposite evidence for
sensitivity of the amygdala to emotional music with eyes open has
also been presented especially with regard to the semantic
structure of music [24,25]. These differences possibly arise from
the nature of stimuli that have been used in studies. The dissonant
or scrambled sounds used by Blood et al.  and Levitin and
Menon  may have evoked less unlikable feelings than
unpleasant real music excerpts in Koelsch et al. [24,25].
The amygdala’s critical role in establishing the emotional
context of sounds is suggested by lesion studies using auditory fear
conditioning in rodents , and by studies in patients with
amygdala excisions investigating the effect of scary music or
unpleasantness of dissonance in music [29,30]. Further support for
the sensitivity of the amygdala to emotional context comes from
few imaging studies using various parameters of fear conditioning
paradigms, meaning of sounds such as crying, and accompanied
unpleasant narrative via words or films [26,31–33]. Here we show
that the amygdala is sensitive to context change derived from
shifting mental set without changing the stimulus complexity. This
further supports our prior interpretation for increased amygdala
activity to music when combined with films, as related to modified
processing style (i.e. mental set) rather than changing mode of
stimulation (i.e. concrete movie and music vs. abstract music
alone) . As previously conjectured, in the presence of negative
music, closing the eyes may be associated with increases in
attention to visceral cues of emotions, as well as appraisal and
conceptual processes. To further examine the way the amygdala is
mediating such processes we looked at its co-activation with
regions most typically proposed to be involved in their operation:
the LC and VPFC, respectively.
The Effect of Eyes Closed on Amygdala’s Connectivity
with the LC
The finding that the amygdala significantly co-activated with
brainstem noradrenergic nuclei such as the LC is consistent with
animal studies showing dense reciprocal connections between the
amygdala and these brainstem nuclei . This connection was
suggested to serve enhanced detection of threat signals through
Figure 6. Correlation analysis for the amygdala ROI. An additional network of co-activation was revealed as during closed eyes in negative
music. Arrows point to main regions of correlated activity with the ROI: (A) LC, (C) NAcc, and (D) anterior temporal gyrus. (B) Co-activation respectively
to the emotional context is depicted. The correlated activity between the amygdala and LC during eyes closed was much stronger in the negative
emotional condition than in the neutral condition.
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vigilance, alertness, and orientation processes [20,35]. In accor-
dance, a recent fMRI study demonstrated increased correlated
activation between the amygdala and the LC when remembering
faces that had been encoded in an emotional negative rather than
neutral context . Intriguingly, another imaging study found
that unaware fear signals were more likely to elicit activity in the
LC than aware fear signals . This sort of finding supports the
idea that the LC serves a relatively low level ‘alarm system’ that
incorporates alerting and orienting signaling to threat-related
stimuli even without full awareness of them. Our current findings
suggest that the amygdala’s connections to adrenergic brainstem
nuclei may mobilize such a low level system under perceptual
context which calls for higher alert (e.g. diminished external visual
information). Though, uniquely here we show that such low level
operation is recruited even for processing relatively complex
cultural based emotional stimuli such as music.
In addition to evidence of increased co-activation between the
amygdala and LC when subjects listened to negative music with
Figure 7. DCM – model and connectivity. (A) directions of the connectivity between the amygdala, PFC and LC during eyes closed. (B) The power
of intrinsic connections indicated by probability and strength. Note the most probable and the strongest connection from the amygdala toward the
PFC. *, p,0.01, **, p,0.001. Blue stars indicate a significant difference between the valences under closed eyes state (p,0.05).
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eyes closed, our causal analysis revealed that this inter-regional
connection was more strongly driven by the amygdala than by the
LC. The LC has been indicated as enabling an appropriate
electrodermal reactivity (i.e. visceral cues of emotions) in the face
of threat, therefore its recruitment by the amygdala under
condition of increased emotionality support a neural basis for
the ‘somatic marker theory’ . However, we did not find
valence selectivity in the modulation effect on this inter-regional
connection under the eyes closed condition suggesting that the
emotional value is not determined by the strength of these
connections. Taken together, our findings propose a picture
whereby closing the eyes in the presence of negative music is
associated with arousal/vigilance modulations exerted through
enhanced amygdala’s drive of brainstem nuclei including the LC.
The LC-amygdala back-projection revealed by the causal analysis
suggests a low level modulation of emotional processing, wherein
brainstem nuclei would facilitate the orienting to affective signals
possibly by providing the amygdala visceral cues of emotion .
Interestingly, the LC did not show increased overall activity to
negative music with eyes closed, consistent with evidence that
arousal modulation may increase effective connectivity between
the LC and attention-related cortical regions without increasing
activity within these regions [20,35].
The Effect of Eyes Closed on Amygdala’s Connectivity
with the PFC
It was suggested that the amygdala is critical to both immediate
signaling of threat in the environment and to slower concept based
cognition regarding negative consequences of such threat .
Our findings support a possibility that the latter is carried out via
amygdala’s connection to the VPFC. The applied causal analysis
suggests that the enhanced effective connectivity between the
amygdala and VPFC is driven by the former when passively
perceiving an emotional stimulus, as in our study (Figure 7).
It was previously claimed that the VPFC is involved in
appraisal-related mechanisms [40–42]. In contrast to the amyg-
dala, the VPFC foci of the closed eyes effect did not show
significant selectivity to the emotional content of music (Figure 2,
bar graphs). The weak selectivity to emotional valence of this
region has been suggested by animal and human lesion studies,
respectively showing that the PFC is not critical for acquiring a
fear conditioned response  and for the association formation of
stimuli with a negative meaning .
However, it is yet possible that the PFC may show selectivity
when appraisal of emotional stimulus is driven by actively
directing focus of attention through task demands. Indeed, we
found that the eyes closed VPFC foci showed a slight trend toward
greater selectivity to emotion when subgroup of our subjects were
instructed to judge the emotionality of musical clips, while the
amygdala was unaffected by this instruction (see Results). This fits
with the proposed role of the VPFC in the regulation of emotional
experience via appraisal mechanisms  and affective decision
[44,46]. Moreover, the results from our causal analysis are
consistent with suggestions that the emotional sensitivity of the
VPFC depends on the modulation of its afferents from the
amygdala . However, possibly by manipulating appraisal
directly the leading direction of the PFC-amygdala connection
might have changed. Indeed, it has been shown that the appraisal
of the emotional valence of stimuli is associated with modulations
of functional connectivity between the amygdala and the VPFC
, and that this functional coupling is diminished in patients
with depression relative to healthy controls during re-appraisal of
negative stimuli .
While our results did not highlight any direct connection
between the LC and the PFC, the former could indirectly
modulate the processing of vigilance-related visceral cues in the
PFC either through its effect on the amygdala or through striato-
cortical loops . Indeed, the ventral striatum receives
glutamatergic afferent input from brain regions that have been
associated with task execution with eyes closed in our study,
including the amygdala, hippocampus, and VPFC. Furthermore,
this interpretation is consistent with the co-activation observed
between the amygdala and the ventral striatum in the whole brain
effective correlation analysis (see Figure 6) indicating a possible
effect of eyes closed on gating mechanisms selective to emotion
value in music .
Segregated PFC Activation with Relation to Mental Set
The close relationship of the VPFC but not DLPFC activation
foci with the amygdala (see Figure 5) is consistent with a proposed
segregation of VPFC and DLPFC functional systems in monkeys
[12,42]. The VPFC which includes the eyes closed activation in
our study (i.e. BA 47 and BA 11) is reciprocally connected with the
amygdala, hippocampus and anterior inferior temporal area and
was proposed to be involved in multimodal internally driven
processing [12,47]. On the other hand, the DLPFC, which
includes the eyes open activation patches in our study, is closely
connected to the low- and high-order visual areas and superior
temporal sulcus (STS) region and was involved in processing
stimuli based on the externally determined features [40–42]. This
dissociation in brain activation pattern related to eyes position is in
line with a study by Hariri et al.  that used an emotional
paradigm in the visual domain. The authors showed that an
implicit emotional processing task was associated with increased
activity in the amygdala/hippocampal region. In contrast, an
explicit emotion processing task requiring the interpretation of
emotions based on acquired knowledge and linking them to
linguistic labels was associated with increased activation in the
DLPFC. Based on these findings we can interpret our results
suggesting that in the eyes closed condition when all visual inputs
become unavailable, compensatory mechanisms for processing
non-visual information from the environment - in this case
mediated by the ventral system - may be automatically recruited.
The VPFC was postulated as important for the identification of
emotional significance of a cue and for the regulation of current
affective states. In contrast, the dorsal system known to be
important in explicit cognitive processes is active during the open-
eyes state when all mechanisms could be involved in the high-
order cognitive tasks (i.e. planning, searching, etc.) . Our
finding reminds another study by Baumgartner et al. , which
compared emotional pictures presented either alone or combined
with congruent emotional musical excerpts. Combining emotional
music with the pictures enhanced their emotional significance
alongside with increased activation in a ventral neural system
comprising the amygdala, brainstem nuclei and ventral regions of
the anterior cingulate cortex and PFC.
Potential Perception-related Confounds
Several perception-related processes could contribute to our
findings. For example, could enhanced emotional experience be
related to intensive imagery induced by the closed eye position?
Our behavioral results make this possibility very unlikely since they
did not reveal a change in concreteness of the sensory experience
while closing the eyes (Figure 1D). Brain wise, the closed eyes state
did not evoke significant increase in activation in exclusively visual
areas as would be expected if the imagery was object selective
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[54,55]. Also no visually-related areas were revealed by correlation
analyses for the eyes closed foci in the VPFC or the amygdala with
whole brain activation (Figure 5). However, increased activation
was seen for eyes closed in the multi-modal regions, such as the
STS and the inferior parietal sulcus (IPS) that were implicated in
mental imagery by others . Though this finding is consistent
with some form of imagery, its exact meaning is unclear. Another
interpretation could be that the eyes closed position modifies
mental schemes related to spatial attention and self-body
reference, both shown to be mediated by the STS and IPS [57–
59]. Further research should explore if and how this perceptual
modification can change emotional experience of music.
Another perceptual effect could be related to visual input
withdrawal with eyes closed. This possibility was tested in the
original experiment by an open eyes condition where subjects
stared at a black screen while listening to the music clips. The lack
of observable effect of negative music on the amygdala during this
condition suggests that the closed eyes effect on the emotional
experience of music cannot be explained by low level visual input
during eyes closed (see Figure 3). However, in an additional
control study, where subjects opened their eyes in complete
darkness, the magnitude of the emotional selectivity of eyes closed
effect in the amygdala seemed weaker (see Figure S2 B in
comparison to Figure 4A). A potential confounding factor was
represented by the fact that half of the subjects in the control study
were repeating the experiment, thus potentially dampening or
otherwise changing the emotional response to the music. This
possibility needs to be tested in greater depth in future studies
using corresponding physiological measures of arousal.
Finally, two additional confounds need to be addressed. First, it
could be argued that subjects found the experience of lying in a
scanner listening to music with eyes closed particularly novel thus
resulting in differences in the amygdala . Our brain results
argue against this likelihood. We base this argument on the fact
that the eyes closed effect was selective for negative music,
whereas subjects experienced neutral music with eyes closed as
well. Second, it could be suggested that a contributing factor to
the closed eyes effects could be related to the lack of gaze control
when eyes are closed. This suggestion seems to be somewhat
unlikely in our study because all conditions with eyes open were
presented under the free viewing and no fixation point was
In summary, our findings support a system-based model in
which context-related variations of processing style induced by
closing the eyes can modify individuals’ emotional experience of
negative stimuli via modified amygdala’s activity. Furthermore,
the amygdala’s connectivity with the LC and VPFC seems to
contribute to such modification, by selectively recruiting,
respectively brain operations related to low level processing of
visceral cues and/or high-level cognitive based processing of
emotions. Our secondary causal analysis further points to the
primacy of the amygdala in driving possible interplays between
these levels of operation under unguided perceived emotionality
in music. The fact that the amygdala is co-operated with regions
involved in both low and high levels of emotional processing
supports the integrative view of the ‘conceptual act model’ in
explaining individual diversities in emotional perception of
stimuli. Speculatively, it can be suggested that disturbed
emotional experiences such as depression and anxiety are evoked
by imbalanced co-operation of these paths of the emotional
system. Future electrophysiological studies can explore whether
these amygdala’s connections are characterized by specific
activation features such as neural synchronization at character-
Materials and Methods
Twenty six healthy subjects participated in the study. Eleven
subjects (5 females, ages 25–41 years) took part in the pre-scan
behavioral measurements. Twelve subjects (6 females, ages 28–36
years) participated in the original fMRI experiment. Six subjects (4
females, ages 23–36 years) participated in the control experiment
(3 of them had also taken part in the original experiment). None of
the subjects had been musical expert; 6 subjects had basic musical
education in youth but did not practice to play during last 15 (and
more) years. All subjects had normal hearing and provided written
informed consent. The IRB committee of the Tel-Aviv Sourasky
Medical Center approved the experimental protocol.
Overall, the music clips consisted of 28 clips, 12 s each. The clips
were un-familiar to the subjects. They contained no human voices
or specific object-related sounds (e.g., a clock ticking, footsteps, etc.).
Initially, the music clips were characterized as positive (joyful; used
in the pre-scan behavioral session only), negative (scary), or neutral
(for examples, listen to Stimuli S1, S2, S3, S4, S5, S6, S7 and S8).
The positive clips were recorded from commercially available CDs
and film sound tracks. The negative clips were composed (cut and
edited) from a horror film sound tracks, from music pieces (using
Wavelab 4.0 from Steinberg, Steinberg Media Technologies,
GmbH, Hamburg, Germany), or created in-house using music
software (Cubase VST 5 from Steinberg, Reaktor 3.0 and Kontakt
1.0 from Native Instruments Software Synthesis, GmbH, Berlin,
Germany). The neutral clips were recorded from commercially
available CDs or created in-house using music software (Cubase
VST 5 from Steinberg, Reaktor 3.0 and Kontakt 1.0 from Native
Instruments Software Synthesis, GmbH, Berlin, Germany). Finally,
the emotional value of the clips was defined based on a pre-scanning
behavioral rating (below).
The average decibel level of all music clips was equalized using
Steinberg Wavelab 4.0. Importantly, all stimuli had similar
acoustical characteristics (all major-minor tonal music), melodic
contour, rhythmic structures and tempo, so, it is highly unlikely
that simply the bottom-up processing of the stimulus character-
istics contributes to activation patterns when contrasting different
conditions. Also, there were no unexpected events in the negative
clips preventing the possibility that the amygdala may be triggered
by them more effectively than by the neutral clips.
The video clips were collected from commercial films. They
were chosen according to the following requirements: (i) clips were
emotionally neutral; (ii) contained no dialogue, and (iii) showed no
widely familiar actors or scenes. The clips have been converted
into the black and white ones and each frame was scrambled using
the Matlab programs. The scrambled clips were too difficult to
follow the movie due to very short presentation of each frame
(40 ms long). Importantly, coupled with emotional music, video
clips could be interpreted as both negative and neutral.
Pre-scan Behavioral Tests of Stimuli
Two pre-scan behavioral sessions were conducted aiming to
evaluate the emotional quality of the musical clips intended for the
fMRI paradigm. The valence levels of musical clips (negative/
neutral/positive) were checked in the first session by presenting 11
naive subjects who did not intend to participate in the fMRI
experiments with 28 different natural musical clips (each 12 s long
and separated by intervals of quiet blank periods). These subjects
were required to estimate the valence (on a scale from 25 to 5)
and arousal (on a scale from 0 to 10) levels of the clips. Ratings for
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each clip were averaged across subjects and were characterized as
either neutral (from 21 to 1), positive (from 1 to 5) or negative
(from 21 to 25). All positive clips and the three negative clips
defined as outliers (i.e., more than 2 STDs away from the mean
rating for negative clips) were excluded from the clip pool. Two
weeks later, the 18 selected clips (9 negative with an average
valence of 22.160.5 and 9 neutral with an average valence of
0.660.4) were presented together with scrambled video clips and
re-evaluated in a similar manner. These results were also averaged
across subjects and four outlier clips were excluded from the final
pool of stimuli.
Original fMRI experiment.
composed of 42 blocks. Specifically, 14 finally pre-selected clips (7
negative and 7 neutral, examples: Stimuli S1, S2, S3, S4, S5, S6,
S7 and S8) were presented to the subjects in 3 different states: eyes
open with a black screen, eyes open with a scrambled video and
eyes closed (Figure 1A). Importantly, all open eyes conditions were
presented under the free viewing - no fixation point was presented
on the screen. To avoid priming effects, the presentation of
negative and neutral clips in relation to eyes position was equally
distributed throughout the experiment. Each clip was 12 s long
and alternated with 6 s blank periods. The experiment lasted a
total of 846 s. The paradigm started with a 15 s black screen blank
period and ended with a similar 12 s period. Verbal instructions to
open or close eyes lasted 3 s each. Six subjects in the original
experiment were requested to covertly perform the valence-rating
of each clip immediately after its presentation (on a scale from 25
to 5; no arousal and abstract level were rated) and six subjects were
scanned while passively listening to music.
paradigm were created. Although the versions consisted of the
exactly the same set of musical and video clips, the order of specific
eye position+music coupling was different across subjects.
Control fMRI experiment.
for the possibility that the difference between eyes closed and eyes
open is due to change in the level of darkness. The experimental
design was identical to the original experiment with the exception
that musical clips (in auditory conditions only, without video) were
presented in complete darkness. Subjects listened to neutral and
negative music with eyes open looking at a black screen, and also
listened to it with eyes closed. Importantly, video clips were not
excluded from this experiment – stimulation was manipulated in
such a way that the projector’s bore was open just before the
condition containing video stimulation and closed immediately
Overall, the experiment was
Two order sequences of the
This study was aimed to control
Behavioral measurements were conducted outside the magnet
on all subjects who participated in the original fMRI experiment
several weeks later. The delay was inspired by several motivations:
(i) we did not measure behavioral performance during the
experiment in order to minimize possible cognitive demand,
which would impact amygdala function as well; (ii) immediate
rating after the scan was not carried out because of subject’s
possible lower sensitivity to stimuli after their repeated presenta-
tion in the magnet. Subjects were presented with the same stimuli
that were used during the scan. The blank periods were extended
to 15 s to allow enough time for their response. The subjects were
required to score the emotional valence (25 to 5), the arousal
feeling (0 to 10) and the abstract level (0 to 10) of all the stimuli. To
ensure that effect of shifting processing style is not related to
increase in imagination, we estimated abstract level of the stimuli.
Subjects were asked to evaluate at what degree they imagine (if so)
the concrete objects or scenes while listening to music. The
average rating was calculated per condition within and across
subjects (Figure 1B–D).
Subjects were scanned in a 1.5 T Signa Horizon LX 8.25 GE
scanner (original experiment) and in a 3 T G3 GE scanner
(Milwaukee, WI, USA) (control experiment) with standard
birdcage head coils. The scanned volume included 25–27 nearly
axial slices of 4 mm thickness and 0 mm gap and covered the
entire cortex. Blood oxygenation level dependent (BOLD) contrast
was obtained with gradient-echo echo-planar imaging (EPI)
sequence (TR=3000 ms, TE=55/35, flip angle=90u, field of
view 24624 cm2).
A whole-brain spoiled gradient (SPGR) sequence was acquired
on each subject to allow accurate cortical segmentation,
reconstruction and volume-based statistical analysis. T1-weighted
high resolution (1.161.1 mm2) anatomical images (124 images,
1.2 mm thickness) of the same orientation as the EPI slices were
acquired to facilitate the incorporation of the functional data into
the 3D Talairach space .
Stimuli were generated on a PC. During scanning, the visual
stimuli were presented to the subjects via an LCD projector (Epson
MP 7200). Subjects viewed them in a titled (,45u) mirror
positioned over their foreheads. The audio stimuli were presented
via the headphones.
fMRI data were analyzed with the BrainVoyager software
package (Goebel, 2000, Brain Innovation, Maastricht, The
Netherlands) and with complementary in-house software. The
first three images of each functional scan were discarded. The
functional images were superimposed on 2D anatomical images
and incorporated into the 3D data sets through trilinear
interpolation. The complete data set was transformed into
Talairach space . Pre-processing of functional scans included
3D-motion correction, linear trend removal, slice scan time
correction, and filtering out of low frequencies up to 3 cycles/
experiment. The cortical surface of each subject was reconstructed
from the 3D SPGR scan. The procedure included segmentation of
the white matter using a grow-region function, the smooth
covering of a sphere around the segmented region, and the
expansion of the reconstructed white matter into the grey matter.
The surface of each hemisphere was then unfolded, cut along the
calcarine sulcus and flattened.
Single subject analysis.
Throughout the statistical analysis, a hemodynamic lag of 3–6 s
was assumed for the model of each subject by maximizing the
extent of the overall activations. A box-car predictor was
constructed for each experimental condition except the blank
period, and the model was independently fitted to the signal of
each voxel. A coefficient was calculated for each predictor using a
A multi-subject analysis was also
performed. The time series of images of all subjects were
converted into Talairach space and z-normalized. For each
subject, the relative contribution of the predictors for each
contrast was estimated separately and then the significance at the
multi-subject level was calculated from the obtained set of values.
Computation of significance values in the activation maps was
based on the individual voxel significance and on the minimum
GLM  statistics were used.
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cluster size of 10 voxels . The multi-subject maps were
obtained using a random effect procedure  and projected on
a single flattened Talairach normalized brain. Statistical levels
were indicated by the color scales. ANOVA was calculated via
StatView 5.0.1 software.
Internal localizer approach.
previously published . Briefly, one subset of the repetitions of
a condition was used to localize the ROI (biased statistically as a
part of the test), while a signal in the complementary subset of the
repetitions (unbiased statistically) was used to evaluate the
activation level. Specifically, two statistical tests were conducted
for each localizer test (e.g. ‘open eyes’ vs. blank period). In each
test, four repetitions of the condition were biased statistically
while the other repetitions of this condition were unbiased
statistically, and activation levels during these blocks were
measured separately. There was no overlap between the
unbiased repetitions of the two tests. Similar activation profiles
in the biased and unbiased subsets can be used as an indication of
a high signal-to-noise ratio. The levels of activation were analyzed
separately for the biased and unbiased data: they were first
averaged for each subject and then averaged across subjects.
Correlation maps analysis.
based on ‘seed’ time courses [66–69]. ‘Seed’ ROIs were
anatomically definedfor each
hemisphere as a cluster with the highest activation level.
Average ‘seed’ time courses were obtained for each subject by
averaging the time series of all voxels in the specific ROI (areas BA
46/9 and BA 47, for example). These average time courses were
used as a GLM predictor to compute a voxel-by-voxel fit
(analogous to linear correlation). Since consecutive fMRI data
points of the regressor are not statistically independent due to the
nature of the hemodynamic response, the fit was evaluated after
removing the auto-regression factor (AR1 model). A second-level
random-effect analysis was applied to determine the brain areas
that showed significant functional activity across subjects. In
contrast to the normally applied procedure in which every
subject’s dataset is fitted with the same design matrix, we used a
different design matrix for each subject, based on the subject’s
actual data (‘seed’ time courses from the same ROIs) so that the
final map reflects regions whose activity is correlated to the activity
in the same ‘seed’ location across subjects.
For the purpose of DCM, fMRI data were reanalyzed
using Statistical Parametric Mapping software package, SPM2
(Wellcome Department of Imaging Neuroscience, London, UK).
DCM is a nonlinear systems identification procedure that uses
Bayesian rules to estimate the influences that one neural system
exerts over another and how this is affected by the experimental
DCM analysis was performed on 14 subjects: 11 out of 12
subjects participated in the original fMRI experiment (one subject
was eliminated due to technical problems) and 3 subjects
participated in the control fMRI experiment but did not
participate in the original experiment (the data sets of 3 subjects
participated in both experiments were taken from the original
experiment). Preprocessing of functional images included motion
correction (realignment to the first volume), slice time correction
(to the middle slice) and normalization to the standard EPI
template of the Montreal Neurological Institute (MNI).
Statistical analysis was relied on a GLM. Events were time-
locked to onset of stimulus presentation and regressors modeling
stimulus events were convolved with a canonical hemodynamic
response function. T-statistical maps were obtained by contrasting
hemodynamic responses during epochs of stimuli presentation of
negative music and neutral music with eyes closed. The analysis of
Details of the approach were
The correlation analysis was
subjectin each cortical
individual subjects was performed at a significance threshold of
The statistical maps of the negative vs. neutral music with eyes
closed contrast were used to define volumes of interest (VOI) for
each subject. The voxel of maximum activation within each VOI
was served as a center of spherical volume (PFC – 6 mm,
amygdala - 3 mm, LC – 2 mm) and defined by the volume of
interest tool integrated in SPM2 (see Table S1 for details on
For each subject a model was defined: an input to the amygdala,
and values of intrinsic connectivity in each direction were
extracted and served for a 2-way ANOVA (factors: valence-
negative, neutral; eyes-closed, open). Probability and strength of
stimuli entering the amygdala were extracted and evaluated in a t-
test (probability – more than 50%, strength – in Hz). Modulating
effects of the negative music and neutral music with eyes closed
were modeled on connections of the amygdala and LC and
connections of the amygdala and PFC.
Activation patterns revealed during eyes closed stimulation (eyes
closed . blank) in the amygdala/anterior hippocampus region for
six different subjects. Compare a dramatic emotion-related effect
revealed for the negative and neutral clips. Regions of interest are
marked by circles. The color scale indicates significance level. L -
left hemisphere, R - right hemisphere, COR - coronal, TRA -
Found at: doi:10.1371/journal.pone.0006230.s001 (6.99 MB TIF)
Activation maps of limbic regions in single subjects.
Multi-subject activation patterns (N=6) obtained by the contrast
‘eyes closed . eyes open’ in the control study are shown on the
transversal view. A strong preferential activation for eyes closed
was found in the amygdala and the anterior hippocampus. The
color scale indicates significance level. L - left hemisphere, R -
right hemisphere, TRA - transversal. (B) Average activation
profiles were obtained in the amygdala (left) and the anterior
hippocampus (right) in the ‘eyes closed . eyes open’ test.
Apertures above the graphs specify stimuli type. The y-axis
denotes an fMRI percent signal change relative to blanks. Note a
significant effect in the amygdala/anterior hippocampus for the
eyes closed state. The asterisk denotes a significant difference
between the eyes open and eyes closed states (p,0.05). Error bars,
Found at: doi:10.1371/journal.pone.0006230.s002 (5.80 MB TIF)
Control experiment - complete darkness effect. (A)
VOI. The location is given as MNI coordinates. The voxel of
maximum activation within each VOI was served as a center of
Found at: doi:10.1371/journal.pone.0006230.s003 (0.10 MB
Volumes of interest used in DCM. Size and location of
and location of regions of interest. The location is given as
Talairach coordinates of the center of the region (X, Y, Z).
Found at: doi:10.1371/journal.pone.0006230.s004 (0.03 MB
Regions of interest used in functional analysis. Size
each subject are presented for un-normalized time-courses. Co-
activation of the amygdala with area BA 47 and co-activation of
the amygdala with area BA 46/9 were computed. Activation in the
BA 47 was obtained in contrast ‘close . open’ for the negative and
neutral stimuli separately. Activation in the BA 46/9 was obtained
Correlation coefficients. Correlation coefficients for
Eyes-Closed and Music
PLoS ONE | www.plosone.org15 July 2009 | Volume 4 | Issue 7 | e6230
in contrast ‘open . close’ for the negative and neutral stimuli
Found at: doi:10.1371/journal.pone.0006230.s005 (0.05 MB
Found at: doi:10.1371/journal.pone.0006230.s006 (1.06 MB AVI)
Negative Stimulus 1
Found at: doi:10.1371/journal.pone.0006230.s007 (1.06 MB AVI)
Negative Stimulus 2
Found at: doi:10.1371/journal.pone.0006230.s008 (1.06 MB AVI)
Negative Stimulus 3
Found at: doi:10.1371/journal.pone.0006230.s009 (1.06 MB AVI)
Negative Stimulus 4
Found at: doi:10.1371/journal.pone.0006230.s010 (1.07 MB AVI)
Neutral Stimulus 1
Found at: doi:10.1371/journal.pone.0006230.s011 (1.06 MB AVI)
Neutral Stimulus 2
Found at: doi:10.1371/journal.pone.0006230.s012 (1.06 MB AVI)
Neutral Stimulus 3
Found at: doi:10.1371/journal.pone.0006230.s013 (1.06 MB AVI)
Neutral Stimulus 4
This study was funded by the Israeli Science Foundation Bikura Program
1440/04 and the Binational Science Foundation (BSF) (TH). We thank M.
Harel for help with the brain reconstructions, Dr. O. Ganor and Dr. E.
Eldar for help with producing the audio stimulation, Dr. G. Avidan and
Dr. T. Siman-Tov for fruitful discussions and comments on the
manuscript. We thank I. Podlipsky for help with the DCM analysis.
Conceived and designed the experiments: YL TH. Performed the
experiments: YL AZ. Analyzed the data: YL LB. Contributed reagents/
materials/analysis tools: YL LB. Wrote the paper: YL DP TH.
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