The middle cingulate cortex and dorso-central insula: A mirror circuit encoding observation and execution of vitality forms

Abstract

Significance
Vitality forms represent the different ways in which actions are performed (e.g., gentle, rude). They express the agent’s attitudes toward others. Previous data indicated that vitality forms of hand actions depend on the dorso-central insula. In the present study, we show that in addition to the insula, the middle cingulate cortex is also involved in hand action modulation. A voxel-based analysis highlighted that voxels showing a similar BOLD signal trend in both action observation and execution are present in both regions. Using a multifiber tractography investigation, we demonstrated that the dorso-central insula and middle cingulate cortex are anatomically connected. These data indicate that the modulation of the parieto-frontal circuit controlling hand actions relies on both the insula and cingulate sectors.

Full text

The middle cingulate cortex and dorso-central
insula: A mirror circuit encoding observation and
execution of vitality forms
G. Di Cesare
a
, M. Marchi
b
, G. Lombardi
a,c
, M. Gerbella
d
, A. Sciutti
a
, and G. Rizzolatti
d,e,1
a
Cognitive Architecture for Collaborative Technologies Unit, Italian Institute of Technology, Genova 16152, Italy;
b
Department of Computer Science, University
of Milan, Milan 20135, Italy;
c
Department of Informatics, Bioengineering, Robotics, and Systems Engineering, University of Genoa, Genoa 16145, Italy;
d
Department of Medicine and Surgery, University of Parma, Parma 43100, Italy; and
e
Istituto di Neuroscienze, Consiglio Nazionale delle Ricerche, Parma43100,
Italy
Contributed by G. Rizzolatti, September 13, 2021 (sent for review June 23, 2021; reviewed by David M. A. Mehler, Marco Tamietto, and Christian Wolf)
Actions with identical goals can be executed in different ways
(gentle, rude, vigorous, etc.), which D. N. Stern called vitality forms
[D. N. Stern, Forms of Vitality Exploring Dynamic Experience in Psy-
chology, Arts, Psychotherapy, and Development (2010)]. Vitality
forms express the agent’s attitudes toward others. In a series of
fMRI studies, we found that the dorso-central insula (DCI) is the
region that is selectively active during both vitality form observa-
tion and execution. In one previous experiment, however, the mid-
dle cingulate gyrus also exhibited activation. In the present study,
in order to assess the role of the cingulate cortex in vitality form
processing, we adopted a classical vitality form paradigm, but
making the control condition devoid of vitality forms using jerky
movements. Participants performed two different tasks: Observa-
tion of actions performed gently or rudely and execution of the
same actions. The results showed that in addition to the insula,
the middle cingulate cortex (MCC) was strongly activated during
both action observation and execution. Using a voxel-based analy-
sis, voxels showing a similar trend of the blood-oxygen-level-
dependent (BOLD) signal in both action observation and execution
were found in the DCI and in the MCC. Finally, using a multifiber
tractography analysis, we showed that the active sites in MCC and
DCI are reciprocally connected.
vitality form network jcingulate jinsula jmirror mechanism j
social interaction
In every action there are two major components: the content
(i.e., the action goal) and the form (i.e., how the goal is
achieved). Whatever its content, actions may be energetic, gen-
tle, rude, but also hesitant or effortless. One can pick up a glass
resolutely or delicately, just as one can shake a hand coldly or
warmly. Specifications of how an action is done do not refer
only to “cold” actions, which are those devoid of an emotional
content, but also to emotions: An individual can experience an
irrepressible or slight sense of disgust or a spurt of explosive or
repressed anger; the face can be contorted in a terrible scowl or
a fleeting grimace.
The vitality forms are therefore the mechanism that modifies
the way in which an action or an emotion is performed or
expressed (its form) by modulating the activity of the basic cir-
cuits controlling the action or the emotion content. As far as
we know, there are no data up to now on the neural basis
through which the vitality form could modulate the expression
of emotions. This despite a rich literature on how emotions can
be conveyed by body expressions, such as facial mimicry and
specific gestures (1). These have been altogether designated as
emotional body language (2). In contrast, there is rich evidence
that the dorso-central insula (DCI) is the neural center that
modulates “cold action.”
The first evidence for this localization was obtained in a
functional MRI (fMRI) study performed by G.D.C. and col-
leagues in collaboration with Stern (3). In this study
participants were presented with video clips showing transitive
and intransitive actions. All actions were performed with two
different vitality forms (gentle or rude). The stimuli were pre-
sented in pairs of consecutive videos, where the “what” of the
observed action and the “how” could be the same or could
change between the video pairs. There were two tasks (what
and how). In the what task, the participants were required to
decide whether the two actions were the same or different,
regardless of their vitality form. In the how task they had to
decide whether the vitality form was the same or different in
the two consecutive videos, regardless of the type of action per-
formed. The most important result turned out to be from the
contrast how vs. what. It revealed a specific activation of the
dorsal-central insula.
In a subsequent fMRI study, G.D.C. et al. (4) confirmed the
role of the dorso-central insula in vitality form coding. They
showed that when an individual observes, plans to perform
(motor imagery), or executes actions conveying vitality forms,
the dorso-central insula exhibits selective activation. Further-
more, conjunction analysis showed that dorso-central insula is
endowed with the mirror mechanism that is a basic brain mech-
anism that transforms sensory representations of others’
Significance
Vitality forms represent the different ways in which actions
are performed (e.g., gentle, rude). They express the agent’s
attitudes toward others. Previous data indicated that vitality
forms of hand actions depend on the dorso-central insula. In
the present study, we show that in addition to the insula,
the middle cingulate cortex is also involved in hand action
modulation. A voxel-based analysis highlighted that voxels
showing a similar BOLD signal trend in both action observa-
tion and execution are present in both regions. Using a mul-
tifiber tractography investigation, we demonstrated that the
dorso-central insula and middle cingulate cortex are anatom-
ically connected. These data indicate that the modulation of
the parieto-frontal circuit controlling hand actions relies on
both the insula and cingulate sectors.
Author contributions: G.D.C., A.S., and G.R. designed research; G.D.C. performed
research; G.D.C., M.M., G.L., M.G., and A.S. analyzed data; and G.D.C., M.G., and G.R.
wrote the paper.
Reviewers: D.M.A.M., Universitatsklinikum Munster; M.T., Universita degli Studi di
Torino; and C.W., University of Heidelberg.
The authors declare no competing interest.
This open access article is distributed under Creative Commons Attribution-
NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).
1
To whom correspondence may be addressed. Email: giacomo.rizzolatti@unipr.it.
This article contains supporting information online at http://www.pnas.org/lookup/
suppl/doi:10.1073/pnas.2111358118/-/DCSupplemental.
Published October 29, 2021.
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behaviour into one’s own motor or visceromotor representa-
tions concerning that behaviour.
Refs. 5–12 confirmed the crucial role of this insula in encod-
ing vitality forms. In a subsequent fMRI study, however, the
same authors found that the cingulate cortex might also be
involved in the encoding of vitality forms (7). In that study, par-
ticipants were asked to identify action types (e.g., stirring cof-
fee, closing a door) by listening to action sounds. Each action
sound was presented gently or rudely (vitality form condition)
or without a vitality form (masked action sounds, control condi-
tion). The results indicated that listening to action sounds con-
veying vitality forms, relative to a control condition activated,
beside the dorso-central insula, the middle cingulate cortex
(MCC). Whereas the activation of the DCI confirmed previous
findings, the activation of the MCC was unexpected.
To ascertain the role of the cingulate cortex in vitality form
processing, we carried out a new fMRI study. We required par-
ticipants to perform two tasks: 1) observe an arm action (obser-
vation task [OBS]) and 2) execute the same action (execution
task [EXE]). In the OBS task, participants observed video clips
showing the right arm of an actor performing actions toward
another actor (e.g., handing over a ball) with a gentle or rude
vitality form (vitality form condition, Fig. 1, A1 and A2 and see
also SI Appendix,SI Methods and Fig. S1) or with no vitality
form (i.e., jerky actions; control condition, Fig. 1, A3). In the
EXE task, participants moved a little box located on a plane
while lying on their chest, as if offering it to the other person,
with a gentle or rude vitality form (vitality form condition) or
with no vitality form (i.e., using jerky movements as a control
condition, Fig. 1, B3).
The main finding of our study is that, in addition to the
insula, the cingulate cortex is selectively involved in the obser-
vation and execution of actions performed with different vitality
forms. Additionally, a voxel-based analysis carried out in these
two cortical regions showed that a large proportion of the most
active voxels are similarly activated during the observation and
execution tasks. Very interestingly, we provided evidence that
the blood-oxygen-level-dependent (BOLD) signal change
extracted in these voxels exhibits a stronger modulation for
rude vitality forms with respect to the gentle ones in both tasks.
Finally, using a multifiber tractography investigation, we dem-
onstrated that the two sites of DCI and MCC controlling vital-
ity forms are anatomically connected.
Results
Cortical Activations during Observation and Execution of Vitality
Forms. The main aim of the present study was to assess the acti-
vation of the cingulate cortex during the OBS and EXE tasks
(see also SI Appendix, Fig. S1). As far as the OBS task is con-
cerned, the contrast vitality forms vs. control (VF OBS vs. CT
OBS) showed that the vitality form condition produced a con-
sistent activation of the left MCC with an extension to the pre-
supplementary motor area (pre-SMA) bilaterally (p value
adjusted with family-wise error correction [p
FWE – corr
] 0.0001,
Ke =1,477 voxels), of the left DCI (p
FWE – corr
0.0001, Ke =
1,486 voxels), of the middle temporal gyrus (left hemisphere:
p
FWE – corr
0.0001, Ke =631 voxels; right hemisphere: p
FWE –
corr
0.0001, Ke =993 voxels; Fig. 2A), and of the right premotor
cortex extending to the inferior frontal gyrus
(p
FWE – corr
0.0001, Ke =2,909 voxels; Fig. 2A). Similarly, for
the EXE task, the contrast vitality forms vs. control (VF EXE
vs. CT EXE) showed that the vitality form condition produced
the activation of the same sectors of the left cingulate cortex
extending to the right side (p
FWE – corr
0.0001, Ke =1,942 vox-
els) and of the insula bilaterally (left hemisphere: p
FWE – corr
0.0001, Ke =520 voxels; right hemisphere: p
FWE – corr
0.002,
Ke =328 voxels; Fig. 2B; for coordinates and statistical values,
see SI Appendix, Table S1).
Conjunction Analysis of Observation and Execution Tasks. The
results of the conjunction analysis of VF OBS vs. CT OBS and
VF EXE vs. CT EXE contrasts revealed the activation of the
MCC with an extension to the pre-SMA (p
FWE – corr
0.0001,
Ke =826 voxels; Fig. 3, A1) and of the left DCI (p
FWE – corr
0.001, Ke =349 voxels; Fig. 3B; for coordinates and statistical
values see SI Appendix, Table S1). These findings imply that the
same voxels located in the MCC and DCI are selectively
involved in the perception and execution of actions endowed
with specific vitality forms (voxels endowed with mirror proper-
ties). Although this analysis showed voxels activated in both
observation and execution tasks it is not informative about the
trend of the BOLD signal between the two tasks. In order to
identify voxels showing a similar trend of the BOLD signal dur-
ing the two tasks (i.e., high signal for VF OBS and high signal
for VF EXE), we carried out a voxel-based analysis. Specifi-
cally, for each voxel we correlated the BOLD signal obtained
for each participant during the observation task (average value)
Fig. 1. Design of the experimental task. First line: the OBS task. Participants observed the right hand of an actor moving an object rightward. Four objects
were used. The observed action could be performed with a gentle (A1)orrude(A2) vitality form, and the task required participants to pay attention to the
action’s vitality form. In the control condition, the participant observed the same action performed in a jerky way (A3). Second line: the EXE task. Participants
held a little box and moved it with a gentle (B1,bluecolor)orrude(B2, red color) vitality form toward the actor in front of them, as shown in the screen. A
cue positioned in the center of the screen indicated when to start the action. During action execution, they saw only the chest of the actor, and the actor’s
hand was outside their field of vision. In the control condition, participants performed the same action in a neutral way (B3). All stimuli in the OBS and EXE
tasks were viewed via digital visors (VisuaSTIM) with a 500,000 px ×0.25 square inch resolution and horizontal eye field of 30°.
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with that obtained during the execution task. This analysis
allowed us to highlight voxels showing a discreet/strong correla-
tion effect between the two tasks i.e., mirror voxels character-
ized by a high correlated activity (HC mirror voxels). Most
importantly, we considered only voxels that showed at least
50% of the explained variance between the two tasks. In this
respect, we decided to use a cutoff correlation value of r>0.7
(coefficient of determination R
2
>=0.49). Results of this anal-
ysis are shown in Fig. 3, A2. Specifically, this picture presents a
grid representing the cingulum and adjacent cortical area (pre-
SMA) subdivided into a series of small squares, each represent-
ing a single voxel. Orange squares indicate voxels selective for
the encoding of vitality forms during both the OBS and EXE
tasks. The results of the correlation analysis showed that 397
out of 826 voxels (48%) in the whole cluster of voxels (cingu-
lum and pre-SMA) showed a significant correlation between
vitality form tasks (VF OBS, VF EXE),112 out of 826 voxels
(13.5%) showed a significant correlation between control tasks
(CT OBS, CT EXE), whereas the remaining voxels (38.5%)
showed a weak correlation between tasks (r<0.7). A further
analysis was restricted to voxels located in the left MCC. This
analysis was carried out applying an inclusive mask of MCC
obtained from a previous fMRI study (7), which showed that
this brain sector was activated during the processing of action
sounds conveying vitality forms. Results of this analysis
revealed that 88 out of 181 voxels (48.6%) in the left MCC
showed a strong significant correlation between vitality form
observation and execution (HC mirror voxels; r>0.7, P<0.05;
Fig. 3, A3). In this brain region, no voxels showed a significant
correlation between control tasks.
Fig. 3, B2 presents voxels located in the insula selective for
the encoding of vitality forms during both the OBS and EXE
tasks. The results of the correlation analysis showed that 147
out of 349 voxels (42.1%) in the whole voxel cluster (insula and
adjacent cortex) showed a significant correlation between
vitality form task (VF OBS, VF EXE), 17 out of 349 voxels
(4.8%) showed a significant correlation between control tasks
(CT OBS, CT EXE), whereas the remaining voxels (53.1%)
showed a weak correlation between tasks (r<0.7). A further
analysis was restricted to voxels located in the left DCI. This
analysis was carried out applying an inclusive mask of DCI
obtained from previous fMRI studies (3–6, 8) which showed
that this brain sector was activated during the processing of
action vitality forms. Results of this analysis revealed that 55
out of 140 voxels (39.2%) showed a strong significant correla-
tion between vitality form observation and execution (HC mir-
ror voxels; r>0.7, P<0.05; Fig. 3, B3). In this brain region, no
voxels showed a significant correlation between control tasks.
Subsequently, from these HC mirror voxels, the BOLD sig-
nal change relative to gentle and rude vitality forms was
extracted to assess their selectivity in vitality form processing.
The comparison between gentle and rude conditions revealed a
significant difference between the BOLD signal change during
the OBS and EXE tasks in both the MCC (Fig. 3, A4) and DCI
(Fig. 3, B4; paired sample ttest, P≤0.05).
White-Matter Tracts Connecting the Insula and Cingulate Cortices.
Connections between the DCI and MCC were found in the left
hemisphere (Fig. 4). In particular, a three-dimensional (3D)
reconstruction of the average tract, obtained using a single tract
from each subject (with 10% threshold), is shown on a 3D brain
template (Fig. 4 Band C).
Discussion
Actions that have the same goal can be performed in different
ways, which Stern termed vitality forms (13, 14). Vitality forms
convey an agent’s internal states and thus play a crucial role in
social interactions. In the last few years, several fMRI studies
have been carried out to identify the neural substrate of vitality
Fig. 2. Brain activations during vitality form processing (A1 and B1). Sagittal and coronal sections showing the activation of the cingulate and insular cor-
tices in the two hemispheres during the direct contrasts VF OBS vs. CT OBS (A2) and VF EXE vs. CT EXE, respectively (B2). These activations are rendered
using a standard Montreal Neurological Institute (MNI) brain template (PFWE <0.05 at cluster level).
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The middle cingulate cortex and dorso-central insula: A mirror circuit encoding
observation and execution of vitality forms
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Fig. 3. Vitality form processing during the OBS and EXE tasks. Encoding of vitality forms in the cingulum (A) and insula (B). Activation maps of the left
cingulum (A1) and insula (B1) resulting from the conjunction analysis of VF OBS vs. CT OBS and VF EXE vs. CT EXE contrasts. These activations are ren-
dered on a standard MNI brain template (PFWE <0.05 at cluster level). Maps of voxels showing a high correlated BOLD activity (r>0.7) during the per-
ception and execution of vitality form actions (hot color) or control actions (cold color) located in the cingulum (whole cluster, orange color, A2 Top),
insula (whole cluster, orange color, B2 Top). Voxels located in the MCC (A3) and DCI (B3) showing a high correlated BOLD activity (r>0.7) during vitality-
forms OBS and EXE tasks (HC mirror voxels). Signal changes in the MCC (A4) and DCI (B4) during the processing of gentle and rude vitality forms. The hor-
izontal line above the columns indicates the comparisons between vitality forms. Asterisks indicate significant differences at *P≤0.05 and ***P≤0.001.
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forms (3–12). These studies have shown that the dorso-central
sector of the insula is the region that is selectively active in both
the perception and expression of vitality forms. Recently, how-
ever, G.D.C. et al. (7) found that listening to action sounds per-
formed gently or rudely produced activation not only of the
DCI but also of the MCC.
To determine whether the MCC is also involved in the encod-
ing of vitality forms, we carried out a new fMRI study with an
experimental paradigm very similar to that used in the basic study
we conducted previously (4), but with a major difference. This dif-
ference consisted of the type of control condition used. In our
previous study, in the control condition participants were asked
to observe or execute an action consisting of the accurate place-
ment of a small ball in a little box. Therefore, the complexity of
the task and the cognitive efforts associated with it (15) could
explain the activation of MCC also in the control condition. In
contrast, in the present study’s execution task, the control condi-
tion consisted of a simple hand action (moving a little box),
whereas the observation task consisted of participants’ observa-
tion of hand actions identical to those in the experimental con-
ditions but performed in a jerky way. These differences in the
control condition may explain why our previous experiment did
not show the activation of the cingulate cortex (see, e.g., Fig. 3).
In fact, it is plausible that the previous experiment’s control con-
dition masked the vitality form effect in the MCC.
In the present study, we also provided evidence that the
MCC is endowed with the mirror mechanism. A voxel-based
analysis showed that the BOLD signal strongly correlated dur-
ing the observation and execution tasks in many voxels located
in the MCC (48.6%) (i.e., HC mirror voxels). An identical anal-
ysis of the DCI showed similar results. The percentage of the
HC mirror voxels located in the DCI was 39.2%. Additionally,
we found that in the HC mirror voxels located in both the
MCC and DCI, the BOLD signal change showed a stronger
activity for rude vitality forms than the gentle ones.
Finally, to identify whether the MCC and DCI sites specifi-
cally related to action vitality forms are anatomically connected,
we carried out a multifiber tractography investigation on the
same group of participants. This analysis showed that these
MCC and DCI sites are linked, forming a vitality form circuit
for hand action. It is well known that the main tractrography
limitation is the possible high false positive rate that has been
observed in some studies (16). However, the robustness of our
results is strongly confirmed by previous tract-tracing studies on
macaque (17, 18). In fact, these connectional studies showed
that in the monkey the sector homolog of the human middle
cingulate cortex is tightly connected with the dorso-central part
of the insula, a part of it known to be involved in modulating
hand movements (19, 20). These data also indicate that the
cingulo-insular circuit here described appears to be well con-
served throughout the primate’s evolution.
Although our study indicates that DCI and MCC are both
involved in encoding vitality form perception and expression,
the fact that MCC is more strongly modulated during the exe-
cution of rude vitality form, relative to the gentle ones, than
DCI suggests that the two areas can have a partial different
functional role, in line with the existing literature.
In this respect it is noteworthy that: First, Craig described
the whole insula as a sensory “interoceptive cortex” that inte-
grates homeostatic, visceral, nociceptive, and somatosensory
inputs (21), through which a representation of the internal
state of the body is generated and, second, according to Kurth
et al. (22), the insula can be subdivided in four main sectors:
the sensorimotor, socioemotional, olfactory–gustatory, and
cognitive ones. The DCI is located in the sensorimotor sector
of the insula and is connected with the parieto-frontal circuit
for reaching/grasping execution and observation (23) as well
as with temporal territories encoding visual and acoustic bio-
logical stimuli (24). The DCI is also involved in processing
the emotional aspects of visual stimuli; in fact a study on a
blindsight patient demonstrated its selective activation for
conscious, and not for unconscious, perception of fearful
bodies (1).
Concerning MCC, in line with our results, previous studies
showed that this cingulate sector is characterized by an evident
motor scaffold. In fact, intracortical stimulation of MCC, car-
ried out on epileptic drug-resistant patients, produced many
typologies of motor acts, including arm, hand, body, and oral
movements. Interestingly, the stimulation of this cingulate sec-
tor produced, before actual movements, the urge to move in
relation to external contingencies (25).
Based on these considerations and on present results, we can
hypothesize that DCI plays an essential role in encoding and
integrating sensory and interoceptive information for generat-
ing the vitality form of the agent, during action execution, and
for encoding those of the observer, during action observation.
In contrast, MCC appears to be more involved in generating
vitality form related to the external contingencies especially in
the case of rude vitality form. However, further studies need to
be carried out to test this hypothesis.
Methods
Sixteen healthy right-handed volunteers (nine females and seven males, mean
age =25.4, SD =2) took part in the fMRI experiment. Due to the COVID-19
pandemic, 14 participants from the same group participated in the second
scanning session, the purpose of which was tocollect diffusion tensor imaging
(DTI) images. All participants had normal or corrected-to-normal visual acuity.
None of them reported a history of psychiatric or neurological disorders or
current use of any psychoactive medications. They gave their written informed
consent to be subjected to the experimental procedure, which was approved
by the Local Ethics Committee of Parma (552/2020/SPER/UNIPR) in accordance
with the Declaration of Helsinki.
Fig. 4. Anatomical connectivity between the insula and cingulum. Activation of the left MCC and DCI resulting from the conjunction analysis of VF OBS
vs. CT OBS and VF EXE vs. CT EXE contrasts (A). White-matter tract connecting the MCC and DCI (two-dimensional [2D] view in B; 3D view in C).
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The middle cingulate cortex and dorso-central insula: A mirror circuit encoding
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Paradigm and Task. The fMRI experiment consisted of two functional runs. In
each run, we presented participants with video clips in two different tasks
(OBS and EXE) and two different conditions (vitality forms, VF; control, CT). In
total, four conditions were presented in independent miniblocks (VF OBS, VF
EXE, CT OBS,CT EXE) in a randomized order (see also SI Appendix,Fig.S1). The
OBS task started with the instruction “observe”and required the participants
to pay attention to the action performed with vitality forms (VF OBS, Fig. 1,
A1) or without vitality forms (CT OBS, Fig.1, A3). The EXE task started with the
instruction “execute”and required the participants to perform the action
themselves (for details see also SI Appendix,Fig.S2). During the EXE tasks, we
presented a static image of the actor seated opposite the obs erver and asked
participants to move a little box toward the actor with vitality forms (gentle
orrude,Fig.1,B1 and B2) or without vitality forms (Fig. 1, B3)bysimplyrotat-
ing the wrist. A cue positioned in the center of the screen indicated when to
start the action, and the color of the edge of the screen indicated the vitality
form to use during the execution of the action (blue color: gentle, Fig. 1, B1;
red color: rude, Fig. 1, B2; gray color: control, Fig. 1, B3). In each video, a fixa-
tion crosswas introduced to control for restrained eye movements.
fMRI Data Acquisition and Analysis. Anatomical T1-weighted and functional
T2*-weighted MR images were acquired with a 3 Tesla General Electrics scan-
ner (details in SI Appendix,SI Methods). After standard preprocessing steps,
data were analyzed using a random-effects model, implemented in a two-
level procedure. In the first level, the fMRI BOLD signal of each participant
was modeled using two general linear models (GLMs),and analysis was carried
out using SPM12 software (the Wellcome Department of Imaging Neurosci-
ence). Subsequently, in the second level, the BOLD signal of all participants
was modeled using two other GLMs, and the group analysis was carried out.
Specifically, in the second-level analysis, the first group analysis was based on
a GLM comprising four regressors (VF OBS, CT OBS, VF EXE, CT EXE) and
enabled us to assess activations associated with each task versus implicit base-
line and activations resulting from the direct contrast between conditions (VF
OBS vs. CT OBS, VF EXE vs. CT EXE; Fig. 2, P
FWE
<0.05 corrected at the clus-
ter level).
In the second group analysis, the BOLD signal was modeled in a GLM com-
prising six regressors (GT OBS, RD OBS, CT OBS, GT EXE, RD EXE, CT EXE) and
enabled us to examine and compare the BOLD signal change during the proc-
essing of gentle and rude vitality forms in the OBS and EXE tasks (details in SI
Appendix,SI Statistical Analysis).
On the basis of the functional maps obtained from the first group analysis,
we carried out a conjunction analysis of the brain activations resulting from
the contrasts VF OBS vs. CT OBS and VF EXE vs. CT EXE (PFWE <0.05 corrected
at the cluster level) to highlight the brainareas active during both the percep-
tion and execution of vitality forms (Fig. 3 Aand B). The results of this analysis
highlighted two regions: the left DCI and the MCC. Subsequently, in these
two regions, for each participant and for each single voxel, we extracted the
BOLD signal change relative to each experimental condition (VF OBS, CT OBS,
VF EXE, CT EXE) using the REX toolbox (https://www.nitrc.org/projects/rex/).
Then, in order to identify and quantify voxels that showed a very similar BOLD
signal change in the OBS and EXE tasks (HC mirror voxels), for each single
voxel, the BOLD signal change obtained during the VF OBS condition was
correlated with that obtained during the VF EXE condition (for details, see
above), and a significant threshold was set at r>0.7 (coefficient of determina-
tion R
2
≥0.49, P<0.05). Finally, considering these voxels located in the DCI
and MCC, in orderto compare the BOLD signal during the processing of differ-
ent vitality forms, we extracted from the functional maps obtained in the
second group analysis the BOLD signal change relative to gentle and rude con-
ditions for both tasks.
Diffusion Data Acquisition and Analysis. In another scanning session, a diffu-
sion spin-echo single shot echo planar imaging sequence with 64 diffusion
directions (effective b-value of 1,000 s/mm
2
), eight images with no diffusion
weight in the anterior–posterior phase encoding direction and eight images
with no diffusion weight in the reverse phase encoding direction were col-
lected from the same participants (details in SI Appendix,SI Methods). Diffu-
sion data were processed using the FMRIB Software Library (FSL) tools (version
5.0.9). After standard preprocessing steps, a further probabilistic tractography
analysis was performed with FSL’s PROBTRACKX tool, testing the connection
of two regions of interest (diameter 12 mm) (DCI coordinates: 32 9 2; MCC
coordinates: 9942).
Data Availability. All study data are included in the article and/or supporting
information.
ACKNOWLEDGMENTS. G.D.C. and A.S. are supported by a starting grant
from the European Research Council (ERC) to A.S. under the European Union’s
Horizon 2020 research and innovation programme. Grant agreement No.
804388, wHiSPER. G.R. is supported by a grant Lombardia 
e Ricerca from the
Lombardia region.
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observation and execution of vitality forms
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