Different amygdala subregions mediate valencerelated and attentional effects of oxytocin in humans
Matthias Gamera,1, Bartosz Zurowskia,b, and Christian BüchelaaDepartment of Systems Neuroscience, University Medical Center Hamburg-Eppendorf, D-20246 Hamburg, Germany; and bDepartment of Psychiatry andPsychotherapy, University of Lübeck, D-23538 Lübeck, Germany

Edited by Mortimer Mishkin, National Institute for Mental Health, Bethesda, MD, and approved April 5, 2010 (received for review January 25, 2010)

The neuropeptide oxytocin enhances the processing of positivesocial stimuli and improves the capacity to effectively attend the eyeregion of conspeciﬁcs. To investigate the neural basis of theseeffects, we combined intranasal oxytocin administration with highresolution functional magnetic resonance imaging in a unique emotion classiﬁcation task. Emotional faces were brieﬂy presented
while controlling for the initial ﬁxation, and measuring subsequenteye movements. Oxytocin had differential effects on the activity of
speciﬁc amygdala subregions. On the one hand, it attenuated activation in lateral and dorsal regions of the anterior amygdala for
fearful faces but enhanced activity for happy expressions, thus indicating a shift of the processing focus toward positive social stimuli. On the other hand, oxytocin increased the likelihood of reﬂexive
gaze shifts toward the eye region irrespective of the depicted emotional expression. This gazing pattern was related to an increase ofactivity in the posterior amygdala and an enhanced functional coupling of this region to the superior colliculi. Thus, different behavioral effects of oxytocin seem to be closely related its speciﬁc
modulatory inﬂuence on subregions within the human amygdala.

|eyes facial expression
perception| functional neuroimaging | neuropeptides | socialIn the past decades, a number of studies have shown that theneuropeptide oxytocin plays an essential role in regulatingsocial behavior in nonhuman mammals. It is crucially involved ininitiating maternal and alloparental behavior in rodents (1) andfacilitates social approach by reducing anxiety and neuroendocrine stress responses (2). Moreover, it enables social bonding involes (3) and enhances the saliency of social information, thereby
strengthening recognition memory for conspeciﬁc animals (4).
Despite these effects of oxytocin on social behavior of rodents andother mammals, this neuropeptide has long been considered a purely
female hormone in humans because of its crucial role in initiating
parturition as well as milk ejection during lactation. However, in
recent years intranasal administration of oxytocin in humans hasrevealed signiﬁcant effects of this neuropeptide on a variety of psychosocial functions (for reviews, see refs. 5 and 6). For example,
oxytocin increases trust (7) and reduces betrayal aversion (8) in human interactions. It has profound effects on behavioral and endocrine stress responses (9, 10) and enhances memory for social
information (11, 12). Moreover, oxytocin increases the human ability
to infer an opponent’s mental state from subtle cues around the eyeregion (13), and it improves the ability to identify fearful faces indynamic displays (14). Because the eye region is a crucial facial
feature for classifying fearful expressions (15), these latter ﬁndings
could be related to an oxytocin-dependent increase in the number
and duration of ﬁxations on the eye region, which was shown ina recent study using neutral faces in free-viewing conditions (16).
With respect to the effects of oxytocin on human brain function,neuroimaging studies have consistently revealed that it reduces
amygdala activity to unpleasant stimuli (17) and to human faces ingeneral (18). Similar suppressive effects have been reported fortrust adaptation in social interactions (8). These ﬁndings wereinterpreted as reﬂecting the neural mechanism of the behaviorally
observed anxiolytic effect of the neuropeptide.
9400–9405 | PNAS | May 18, 2010 | vol. 107 | no. 20

Taken together, these ﬁndings suggest that oxytocin increases
gaze on the eye region while it decreases amygdala activity whenobserving human faces. Such ﬁndings conﬂict, however, with a recent study revealing that increased rather than decreased amygdala
activation predicted reﬂexive gaze changes toward the eye region(19). This observation precisely accounts for the clinical ﬁndings ofreduced spontaneous ﬁxations on the eye region and a failure to
identify fearful expressions in a patient with complete bilateral
amygdala damage (20, 21). Thus, the behavioral effects of oxytocin
in increasing gaze on the eye region (16) seem to be at odds with thereported reduction of amygdala activity in other studies (17, 18). A
possible explanation for this discrepancy is the introduction of large,
partial-volume effects by the low spatial resolution of standard
fMRI acquisition schemes in comparison with the small size of individual amygdala nuclei that may respond differentially to oxytocin
treatment. This notion is in agreement with data in voles (22)showing a regionally speciﬁc distribution of oxytocin receptors
within the amygdala. Moreover, a functional dissociation of amygdala subregions during social conditioning has recently been observed in humans (23). These ﬁndings could signify that one
amygdala subregion shows an oxytocin-dependent reduction of activity for negative stimuli, possibly reﬂecting the anxiolytic effect of
the neuropeptide, whereas a different subregion is up-regulated byoxytocin, thereby facilitating gaze orienting toward the eye region ofemotional faces.
To examine these hypotheses in detail, we carried out a doubleblind placebo-controlled study using high-resolution functional
magnetic resonance imaging (fMRI). Participants were instructed
to classify brieﬂy presented negative (fearful), positive (happy), or
neutral facial expressions as quickly and accurately as possible. To
examine the effect of oxytocin on gaze-orienting behavior, stimuli
were unpredictably shifted either downward or upward on eachtrial, thus manipulating whether participants initially ﬁxated on theeye or mouth region. Stimuli were brieﬂy presented (150 ms) whilemeasuring brain activity with high-resolution fMRI (2 × 2 × 2 mm3).
Furthermore, on-line eye tracking was used to identify reﬂexive
gaze changes that were triggered by these faces but occurred after
stimulus offset (19). The pharmacological manipulation was realized in a double-blind between-subjects design with 46 participants either receiving a single intranasal dose of 24 IU oxytocin(Syntocinon spray; Novartis; three puffs per nostril, each with 4 IU
oxytocin) or a placebo spray that contained all inactive ingredients
except for the neuropeptide (7). This setup allowed us to examine
modulatory effects of oxytocin on valence processing as well as on
gaze-orienting behavior with high anatomical resolution.

Author contributions: M.G. and C.B. designed research; M.G. and B.Z. performed research;
M.G. analyzed data; and M.G., B.Z., and C.B. wrote the paper.
The authors declare no conﬂict of interest.
This article is a PNAS Direct Submission.
See Commentary article on page 9033.
1

To whom correspondence should be addressed. E-mail: m.gamer@uke.uni-hamburg.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1000985107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1000985107

SEE COMMENTARY

Results
Behavioral Data. For each participant and condition, we measured

the proportion of correct responses as well as the mean reaction
time in the emotion classiﬁcation task. Overall, participants inboth groups were highly accurate in classifying the depicted
emotional expressions (placebo: mean ± SD = 96.6 ± 0.8%;
oxytocin: mean ± SD = 97.0 ± 0.5%; see Table 1), and none of
the experimental factors had a statistically signiﬁcant inﬂuenceon classiﬁcation accuracy.
As can be seen from Fig. 1A, response times were longer for
fearful faces [main effect emotion: F(2, 88) = 6.97, ε = 0.86, P =0.003]. However, we also obtained a signiﬁcant interaction ofemotion and initial ﬁxation [F(2, 88) = 12.20, ε = 1.00, P <0.001], indicating that participants were faster in classifying the
depicted expression when they initially ﬁxated diagnostically
relevant features (i.e., the eye region for fearful faces and themouth for happy facial expressions) (15). All other effects did
not reach statistical signiﬁcance.

Imaging Data: Valence Effects. In a number of previous studies it was
shown that fearful faces elicit larger amygdala activation than
happy faces (24, 25). To examine whether this differential response is altered by oxytocin, we calculated the interaction effectof drug (placebo vs. oxytocin) and facial expression (fearful vs.
happy) on the imaging data. In the placebo group, this analysis
conﬁrmed previous reports and revealed a signiﬁcantly enhanced
response to fearful faces in the left amygdala. Interestingly, thispattern was reversed under oxytocin (Fig. 2). This effect was speciﬁc, because additional analyses contrasting both groups acrossall facial expressions conﬁrmed that oxytocin had no generalsuppressing or up-regulating effect on amygdala responses.
Imaging Data: Gaze-Related Effects. As described previously, weobserved that oxytocin increased the number of ﬁxation changes
toward the eye region. To determine the neural correlate of thiseffect, we estimated the interaction of drug (placebo vs. oxytocin)
and initial ﬁxation (eyes vs. mouth) in the imaging data. This
Table 1. Proportion of correct emotion classiﬁcation in theplacebo and the oxytocin group as a function of the depictedfacial expression and the initial ﬁxation
Placebo

Oxytocin

Emotion

Eyes

Mouth

Eyes

Mouth

FearfulHappy
Neutral97.2 (0.8)
97.1 (1.6)
94.4 (1.9)

97.0 (0.9)
95.9 (2.5)
98.2 (0.5)

95.5 (1.5)
98.3 (0.5)
96.6 (1.1)

96.6 (1.0)
98.1 (0.6)
96.9 (0.7)

All ﬁgures are percentages. Values in parentheses indicate SEM.

Gamer et al.

Fig. 1. Behavioral responses and eye movements in the oxytocin and theplacebo group as a function of facial expression and initial ﬁxation. (A)Response times. (B) Proportion of ﬁxation changes targeting the other major
facial feature. Error bars indicate SEM.

analysis revealed a robust bilateral interaction effect in the superior colliculi that resembled the observed gazing pattern (Fig. 3).
The superior colliculi can be considered an output structure thatis highly relevant for initiating saccades (26). Thus, the aforementioned interaction effect seems to be closely related to the
behavioral ﬁnding of an increased number of ﬁxation changes
toward the eye region in the oxytocin group. To get further insight
into how other brain regions might be coupled to the superior
colliculi, we classiﬁed all stimuli according to the participant’s
gazing behavior. Subsequently, we contrasted stimuli triggeringﬁxation changes toward the eye region with trials where participants continued ﬁxating on the mouth. This analysis revealed
a signiﬁcant cluster in the right posterior amygdala showing
a larger response on trials with ﬁxation changes toward the eyeregion in the oxytocin as compared with the placebo group (Fig. 4).
To determine whether the enhanced gaze-related response of
the amygdala in the oxytocin group is functionally related to theactivation of the superior colliculi, we carried out a physiophysiological interaction analysis. First, we extracted the meantime series for each participant from the left superior colliculus(x = −8, y = −30, z = −9), which showed the largest interaction
effect of drug and initial ﬁxation. These time series were thenused to determine the correlation of this seed region with thewhole measured brain volume. Finally, we searched for regions
showing an enhanced functional coupling with the superior colliculus in the oxytocin as compared with the placebo group. The
analysis revealed a signiﬁcant cluster in the right posterior
amygdala (Fig. 5), which largely overlaps with the gaze-related
response in the oxytocin group (Fig. 4). Thus, oxytocin increased
the functional coupling of amygdala and superior colliculus.
Discussion
In the placebo as well as the oxytocin group, participants were
faster in recognizing fearful expressions when initially looking at
the eye region, and identiﬁed happy faces with shorter latencies
when initially ﬁxating the mouth, which is in agreement with the
distribution of diagnostic facial features (15). Correspondingly,
gaze changes toward the eye region of fearful faces occurred more
frequently than for happy ones, and the opposite pattern was observed for ﬁxation changes toward the mouth (19). Although
PNAS | May 18, 2010 | vol. 107 | no. 20 | 9401

NEUROSCIENCE

Eye Movement Data. From the acquired eye-tracking data, we
calculated whether a ﬁxation change occurred within a period of
1,000 ms following stimulus offset. Subsequently, the proportionsof reﬂexive gaze changes from the eye region toward the mouthand from the mouth region toward the eyes were determinedbased on whether participants initially ﬁxated on the eyes ormouth, respectively (19).
Across both groups, reﬂexive gaze changes followed the distribution of diagnostically relevant features in the face [interaction ofemotion and initial ﬁxation, F(2,88) = 13.94, ε = 1.00, P < 0.001].
That is, gaze changes toward the eye region were more likely for
fearful and neutral faces than for happy facial expressions thattriggered more gaze changes toward the mouth (Fig. 1B). In addition, we observed a signiﬁcant interaction of drug and initial
ﬁxation [F(1,44) = 4.35, P = 0.043], indicating that oxytocin increased the likelihood of gaze changes toward the eyes irrespectiveof the depicted facial expression.

Fig. 2. Amygdala regions showing a signiﬁcant interaction of group (placebo, oxytocin) and emotional expression (fearful, happy). (Right) Statistical map(coronal and axial plane) of the interaction effect revealing two clusters in the left amygdala [dorsal cluster, peak voxel: x = −27, y = −2, z = −17 mm, volume =121 mm3, t(44) = 4.37, P = 0.017, FWE corrected; and ventral cluster, peak voxel: x = −23, y = −3, z = −28 mm, volume = 75 mm3, t(44) = 3.88, P = 0.058, FWE
corrected]. (Left) Contrast estimates of these clusters, with error bars representing SEM. The cluster in the right amygdala did not reach statistical signiﬁcance[peak voxel: x = 23, y = −1, z = −19 mm, volume = 16 mm3, t(44) = 2.74, P = 0.004, uncorrected].

oxytocin did not directly affect the efﬁciency of processing emotional faces per se, the neuropeptide increased the proportion ofﬁxation changes toward the eyes across all expressions. This
resembles a possible mechanism through which oxytocin enhances
the human ability to infer the mental state of others from subtle
cues around the eyes (13), and improves the detection of fear infaces (14), because both skills critically depend on elaborately
scanning the eye region (21).
The effect of oxytocin on gazing behavior was related to an
enhanced activation of the right posterior amygdala, most likely
the basal nucleus (27), on trials where gaze changes toward the eyeregion occurred. The anatomical location of this activation closely
resembles a recent ﬁnding of this region being predictive for theoccurrence of reﬂexive gaze movements toward fearfully widenedeyes (19). The current study further demonstrated a functional

connection of this amygdala subregion with the superior colliculi,
which increased when participants received oxytocin as comparedwith placebo. Animal research indicates that the superior colliculi
are highly relevant for covert shifts of attention as well as for
initiating saccades toward behaviorally relevant targets (26).
Furthermore, collicular lesions disrupt reﬂexive attention and eye
movements in humans (28). Amygdala lesions, however, impair
reﬂexive gaze orienting toward the eye region (21) and result in an
inadequate use of gaze cues pointing to behaviorally relevant
locations (29). Thus, both regions, the amygdala and the superior
colliculi, are implicated in redirecting attention toward sociallyrelevant locations in the visual ﬁeld. The current data indicate thatspeciﬁcally the basal nucleus of the amygdala might be crucial
for assigning salience to speciﬁc facial features in the visual periphery (21). Subsequently, the reﬂexive orienting of attention

Fig. 3. Regions showing an enhanced response in the oxytocin as compared with the placebo group when participants initially looked at the mouth region.
(Upper) Statistical map (axial and coronal plane) of this interaction revealing a robust bilateral effect in the superior colliculi [left cluster, peak voxel: x = −8,
y = −30, z = −9 mm, volume = 440 mm3, t(44) = 4.03, P < 0.0001, uncorrected; right cluster, peak voxel: x = 6, y = −33, z = −8 mm, volume = 116 mm3, t(44) =3.48, P < 0.001, uncorrected]. (Lower) Contrast estimates of the peak voxel in the left superior colliculus, with error bars representing SEM.

9402 | www.pnas.org/cgi/doi/10.1073/pnas.1000985107

Gamer et al.

toward these stimuli is enabled by modulating activity in the superior colliculi. Importantly, activity in this circuit seems to be upregulated by oxytocin, thus improving the processing of sociallyrelevant information from the eye region of another person (13).
In addition to the gaze-related effect of oxytocin on amygdala
activity, we observed a valence modulation in an anatomicallydistinct region within the left anterior amygdala, with fearful faces
eliciting larger activity than happy expressions in the placebogroup. This effect, which is in line with previous studies showingthat the amygdala is sensitive to aversive stimuli (24, 25), was foundin two different amygdala subregions that correspond to the corticomedial nuclear group as well as to the lateral amygdaloid nucleus (27). However, oxytocin did not only reduce amygdala
activity for threatening stimuli in these regions as has beenreported (17), but additionally increased responses to happy facial
expressions in the present study. Recent accounts on amygdalafunction have focused on a more general role of this structure in
identifying salient stimuli in the environment instead of speciﬁcallyprocessing aversive information (30, 31). Animal studies suggestthat the lateral and basal nuclei of the amygdala receive highly
processed sensory information (32) that enables detection of suchbiologically relevant stimuli in the environment. The central
amygdaloid nucleus (part of the corticomedial nuclear group),with its modulatory inﬂuences on sympathetic arousal, was linked
to vigilance and attention (33). These subregions of the amygdala
might thus be involved in detecting whether a given stimulus isFig. 5. Amygdala regions showing an enhanced functional coupling with the left superior colliculus in the oxytocin as compared with the placebo group.
(Left) Signiﬁcant cluster in the right posterior amygdala on a coronal plane [peak voxel: x = 29, y = −9, z = −15 mm, volume = 179 mm3, t(44) = 3.99, P = 0.037,
FWE corrected]. (Right) Mean correlation coefﬁcients (values were Fisher z-transformed before averaging) between the activity in both regions as a functionof group. Error bars indicate SEM.

Gamer et al.

PNAS | May 18, 2010 | vol. 107 | no. 20 | 9403

SEE COMMENTARYNEUROSCIENCE

Fig. 4. Amygdala regions reﬂecting gaze preferences for the eye region ona trial-by-trial basis. (Right) Coronal plane shows an enhanced amygdala
response in the oxytocin as compared with the placebo group when participants shifted their gaze toward the eye region [peak voxel: x = 28, y = −9,z = −14 mm, volume = 112 mm3, t(44) = 4.71, P = 0.007, FWE corrected]. (Left)
Cluster location on sagittal and axial planes. The lines labeled −1 depict the yposition of the valence-related oxytocin effect in the left amygdala that isshown in Fig. 2.

biologically relevant, and allowing the organism to adjust its current level of arousal appropriately.
Typically, negative stimuli have higher behavioral relevancethan positive stimuli, which could account for the frequently observed enhanced amygdala activation for aversive stimuli. However, the modulatory effect of oxytocin on amygdala activity in thecurrent experiment might indicate that oxytocin triggers a shift ofthis processing focus from negative to positive social stimuli tofacilitate approach behavior (34). In humans, it was demonstrated
that oxytocin reduces misclassiﬁcations of positive emotions (35)and enhances the encoding of happy facial expressions (11). Furthermore, it increases trust in human interactions (7) and facilitates the recognition of positive stimuli conceptually associatedwith sexuality, bonding, and social relationships (36). These behavioral ﬁndings indicating an enhanced processing of positivesocial information while reducing the evaluation of negative
stimuli may result from the modulatory effect of oxytocin on
amygdala function that was found here.
Recent reviews on oxytocin functions in humans have proposedthat this substance might be used to support the treatment of
mental disorders that are characterized by marked deﬁcits in social
behavior (5, 37). For example, patients with autism spectrum
disorder have deﬁcits in social interactions as well as impairments
in understanding the mental states of others. Moreover, it was
found that these patients show a hypoactivation of the amygdala(38, 39) as well as behavioral difﬁculties in identifying facial
expressions (40). These ﬁndings may be related to abnormal ﬁxation patterns and gaze aversion when scanning facial stimuli (41).
Our current data suggest that oxytocin might improve these
symptoms by enhancing amygdala activity, thereby facilitatinggaze shifts toward the eye region. In fact, recent studies showedthat a single intranasal dose of oxytocin improved emotion recognition from the eye region in young people with autism spectrum
disorders (42), and it increased patients’ gazing time on the eyeregion in free-viewing conditions (43).
Social anxiety disorder is another clinical condition that might
beneﬁt from oxytocin treatment. Patients with this disorder showenhanced amygdala reactivity to faces depicting negative emotions(44) and a lower number of ﬁxations on the eye region (45).
However, in contrast to autism spectrum disorders, patients withsocial anxiety disorder typically have a preserved ability to identify
facial expressions (46). Thus, their gaze aversion seems to be related more to the enhanced stress and anxiety of being evaluatedby others; correspondingly, these patients tend to focus on negative information in social situations (47). As we showed that oxytocin may facilitate a shift of the processing focus from negative topositive social stimuli by suppressing amygdala activity to fearfulfaces while enhancing it for happy expressions, a supporting ad-ministration of this neuropeptide in the treatment of social anxiety
might be helpful. First attempts of using oxytocin to improve exposure therapy have been made (48), but future research will haveto examine whether positive effects during treatment generalize toreal-life conditions.
We have shown that oxytocin has signiﬁcant effects on anatomically distinct amygdala regions associated with valence processing and gaze orienting. These data complement recent ﬁndings
from monkeys (49) and humans (23) showing a dissociation ofactivity within the amygdala, with separate subregions subserving
different aspects of face processing. In the current study, oxytocin
increased the likelihood of reﬂexive gaze shifts toward the eyeregion of faces depicting different emotions. This gaze pattern was
related to an increase of activity in the basal amygdaloid nucleus
and an enhanced functional coupling of this region to the superior
colliculi. Furthermore, oxytocin attenuated responses to fearfulexpressions while enhancing them for happy faces in the corticomedial part of the amygdala and the lateral amygdaloid nucleus.
These speciﬁc effects of oxytocin on amygdala function may underlie the diverse modulatory effects of this neuropeptide on human social behavior (5, 6) and may be relevant for novel attempts
at using oxytocin for the treatment of mental disorders (50).
Materials and Methods
Subjects. Forty-six healthy, right-handed, male volunteers (age mean ± SD =25.0 ± 3.7 years) participated in this study that was approved by the ethics
committee of the medical faculty of the University of Rostock. Exclusion criteria for participation were a history of neurological or endocrine disease,
medication, and drug or alcohol abuse. Furthermore, subjects were instructed
to abstain from alcohol and nicotine for 12 h before the experiment. All participants gave written informed consent and were paid for participation.
Experimental Protocol. In a double-blind, placebo-controlled study design,
participants were assigned either to the experimental group that received
a single intranasal dose of 24 IU oxytocin (Syntocinon spray; Novartis; threepuffs per nostril, each with 4 IU oxytocin) or to the control group, which received
a placebo spray containing all inactive ingredients except for the neuropeptide.
It was previously shown that intranasally applied neuropeptides cross the
blood–brain barrier (51) and therefore allow for studying oxytocin effects on
brain function (37). Because central nervous oxytocin levels reach a plateau
approximately 40 min after substance administration, participants received
oxytocin or placebo 45 min before fMRI scanning in the present study. The
whole functional neuroimaging session lasted no longer than 45 min.
To control for nonspeciﬁc effects of oxytocin, two state questionnaires
were completed: (i) after arriving at the laboratory, (ii) directly before the
MRI scanning (∼40 min after drug application), and (iii) after participants
left the MRI scanner. Neither the initial values of these questionnaires norchanges during the experiment differed between the oxytocin and theplacebo group (SI Results, Fig. S1).
Emotion Classiﬁcation Paradigm. The experimental task was based on a fully
crossed 3 × 2 within-subjects design with the factors emotion and initial ﬁxation. Male and female faces unambiguously depicting neutral, fearful, andhappy expressions were selected from several established stimulus sets (SIMaterials and Methods). A different sample of 72 individual faces (12 males
and 12 females for each expression) was randomly selected from this pool of
pictures for each participant. These faces were then presented once in a randomized sequence in each of three experimental sessions. A trial started witha ﬁxation cross (2 s) followed by the presentation of the face (150 ms) followed
by a blank screen (1,850 ms) and another ﬁxation cross for a randomly chosen
period of 2–9 s. To precisely control for the initial ﬁxation, half of the male andfemale stimuli within each emotional expression were unpredictably shifted
either downward or upward on each trial such that the eyes or the mouth
appeared at the location of the ﬁxation cross. Participants were instructed toclassify the depicted emotional expression as quickly and accurately as possible
by pressing the corresponding key on a button box with the right hand. Before
scanning, participants were trained on the task using a different set of six faces
to familiarize them with the button assignment.
Data Acquisition. Blood oxygenation level-dependent functional images andeye movement data were acquired throughout the experiment. Stimuli were
projected onto a translucent screen using a video projector, and subjects

9404 | www.pnas.org/cgi/doi/10.1073/pnas.1000985107

viewed this screen via a special mirror that was mounted to the head coil. Thisdichroic reﬂector allowed infrared light to pass while reﬂecting the visible
light from the video projector. The 60-Hz MRI-compatible eye-tracking
camera (NordicNeuroLab) was hidden behind the mirror and mounted directly in front of the right eye of the participant.
Functional imaging was performed on a 3-Tesla whole-body MR scanner(Trio; Siemens) equipped with a 32-channel head coil. Thirty-ﬁve transverse
slices (slice thickness, 2 mm; no gap) were acquired in each volume covering the
temporal lobe and occipital and orbitofrontal cortex. A T2*-sensitive gradientecho-planar imaging (EPI) sequence was used (repetition time [TR] = 2,240 ms;
echo time [TE] = 30 ms; ﬂip angle = 80°; FOV = 208 × 208 mm; in-plane resolution 2 × 2 mm). Additionally, isotropic high-resolution (1 × 1 × 1 mm3)
structural images were recorded using a T1-weighted coronal-oriented
MPRAGE sequence with 240 slices.
Data Analysis: Behavior. Details on the eye movement analysis can be found in
SI Materials and Methods. In short, we determined the proportion of ﬁxation changes (>0.5°) toward the other major facial feature that were triggered by the stimulus but occurred after stimulus offset. That is, when theeyes were presented at the position of the ﬁxation cross, we determined the
proportion of downward ﬁxation changes toward the mouth, and when themouth followed the ﬁxation cross, we calculated the corresponding proportion of upward ﬁxation changes toward the eyes.
The proportion of ﬁxation changes, as well as the behavioral data (proportion of correct emotion classiﬁcations and response times), were analyzed
with a series of 2 × 3 × 2 ANOVAs using drug (oxytocin vs. placebo) asa group factor, and emotion and initial ﬁxation a within-subjects factors.
Where appropriate, the Huynh–Feldt procedure was applied to correct forpotential violations of the sphericity assumption. A rejection region of P <0.05 was used for these statistical tests.
Data Analysis: fMRI. Statistical parametric mapping (SPM8, Wellcome Department of Imaging Neuroscience, London) was used for preprocessing andanalyzing the imaging data. A narrow 4-mm full-width at half maximum(FWHM) isotropic Gaussian smoothing kernel was used during preprocessing
to optimize the detection of small activation foci within the amygdala (fordetails, see SI Materials and Methods).
Three different random-effects analyses were carried out. First, to examinechanges in brain activation as a function of the experimental manipulations,
we constructed a design matrix for each subject by modeling the onset of theface presentation as separate regressors for all six combinations of emotion(fearful, happy, neutral) and initial ﬁxation (eyes, mouth) in each session. The
ﬁxation period between the events served as baseline. Regressors wereconvolved with the hemodynamic response function, and one contrast was
speciﬁed to reveal whether oxytocin altered the frequently reported larger
amygdala activation for fearful as compared with happy facial expressions(24, 25). A second contrast was computed to compare brain activation between the initial ﬁxations (eyes vs. mouth). Finally, statistical maps for bothcontrasts were separately subjected to second-level analyses (t tests) tocompare the responses between the oxytocin and the placebo group.
A second analysis focused on the correlation of brain activity and gazing
behavior on a trial-by-trial basis. For this analysis, trials were classiﬁed into
one of four categories using the acquired eye-tracking data. The ﬁrst category speciﬁed ﬁxation, i.e., no gaze change occurred in a period of 1,000 ms
following stimulus offset. Trials with gaze changes toward the other major
facial feature represented the second category. The remaining trials werethen classiﬁed according to whether a gaze change into another direction
occurred or whether a given trial could not be analyzed (e.g., due to eyeblinks). Table S1 depicts the proportion of trials that fell into each of thesefour trial categories as a function of the experimental manipulations. This
classiﬁcation was used to specify a design matrix for each subject by modeling the onset of the face presentation as separate regressors for all combinations of emotion, initial ﬁxation, and gazing behavior. These regressors
were convolved with the hemodynamic response function. To reveal brain
regions mediating the increased likelihood of reﬂexive gaze changes toward
the eye region in the oxytocin group (Results), we calculated difference
maps for each subject individually, contrasting trials with and without gazechanges toward the eye region. As we did not ﬁnd an emotion-speciﬁc effect of oxytocin on gazing behavior (Results), all emotional expressions werepooled in these contrast maps. On the second level, a t test was performed
to determine differences between the oxytocin and the placebo group.
The third analysis was carried out to test for drug effects on the functional
coupling between the superior colliculi and the amygdala. For this aim, we
extracted the mean time series for each subject from a seed voxel in the left
superior colliculus at x = −8, y = −30, z = −9 (for the identiﬁcation of thisGamer et al.

1. Ross HE, Young LJ (2009) Oxytocin and the neural mechanisms regulating social
cognition and afﬁliative behavior. Front Neuroendocrinol 30:534–547.
2. Bartz JA, Hollander E (2006) The neuroscience of afﬁliation: Forging links between
basic and clinical research on neuropeptides and social behavior. Horm Behav 50:
518–528.
3. Hammock EAD, Young LJ (2006) Oxytocin, vasopressin and pair bonding: Implications
for autism. Philos Trans R Soc Lond B Biol Sci 361:2187–2198.
4. Choleris E, Clipperton-Allen AE, Phan A, Kavaliers M (2009) Neuroendocrinology ofsocial information processing in rats and mice. Front Neuroendocrinol 30:442–459.
5. Heinrichs M, von Dawans B, Domes G (2009) Oxytocin, vasopressin, and human social
behavior. Front Neuroendocrinol 30:548–557.
6. Neumann ID (2008) Brain oxytocin: A key regulator of emotional and social
behaviours in both females and males. J Neuroendocrinol 20:858–865.
7. Kosfeld M, Heinrichs M, Zak PJ, Fischbacher U, Fehr E (2005) Oxytocin increases trust in
humans. Nature 435:673–676.
8. Baumgartner T, Heinrichs M, Vonlanthen A, Fischbacher U, Fehr E (2008) Oxytocin
shapes the neural circuitry of trust and trust adaptation in humans. Neuron 58:
639–650.
9. Heinrichs M, Neumann I, Ehlert U (2002) Lactation and stress: Protective effects ofbreast-feeding in humans. Stress 5:195–203.
10. Heinrichs M, Baumgartner T, Kirschbaum C, Ehlert U (2003) Social support andoxytocin interact to suppress cortisol and subjective responses to psychosocial stress.
Biol Psychiatry 54:1389–1398.
11. Guastella AJ, Mitchell PB, Mathews F (2008) Oxytocin enhances the encoding of
positive social memories in humans. Biol Psychiatry 64:256–258.
12. Rimmele U, Hediger K, Heinrichs M, Klaver P (2009) Oxytocin makes a face in memory
familiar. J Neurosci 29:38–42.
13. Domes G, Heinrichs M, Michel A, Berger C, Herpertz SC (2007) Oxytocin improves“mind-reading” in humans. Biol Psychiatry 61:731–733.
14. Fischer-Shofty M, Shamay-Tsoory SG, Harari H, Levkovitz Y (2010) The effect of
intranasal administration of oxytocin on fear recognition. Neuropsychologia 48:
179–184.
15. Smith ML, Cottrell GW, Gosselin F, Schyns PG (2005) Transmitting and decoding facial
expressions. Psychol Sci 16:184–189.
16. Guastella AJ, Mitchell PB, Dadds MR (2008) Oxytocin increases gaze to the eye regionof human faces. Biol Psychiatry 63:3–5.
17. Kirsch P, et al. (2005) Oxytocin modulates neural circuitry for social cognition and fearin humans. J Neurosci 25:11489–11493.
18. Domes G, et al. (2007) Oxytocin attenuates amygdala responses to emotional faces
regardless of valence. Biol Psychiatry 62:1187–1190.
19. Gamer M, Büchel C (2009) Amygdala activation predicts gaze toward fearful eyes. J
Neurosci 29:9123–9126.
20. Adolphs R, Tranel D, Damasio H, Damasio A (1994) Impaired recognition of emotion
in facial expressions following bilateral damage to the human amygdala. Nature 372:
669–672.
21. Adolphs R, et al. (2005) A mechanism for impaired fear recognition after amygdala
damage. Nature 433:68–72.
22. Insel TR, Shapiro LE (1992) Oxytocin receptor distribution reﬂects social organizationin monogamous and polygamous voles. Proc Natl Acad Sci USA 89:5981–5985.
23. Davis FC, Johnstone T, Mazzulla EC, Oler JA, Whalen PJ (2010) Regional response
differences across the human amygdaloid complex during social conditioning. Cereb
Cortex 20:612–621.
24. Morris JS, et al. (1998) A neuromodulatory role for the human amygdala in processing
emotional facial expressions. Brain 121:47–57.
25. Fusar-Poli P, et al. (2009) Functional atlas of emotional faces processing: A voxelbased meta-analysis of 105 functional magnetic resonance imaging studies. J
Psychiatry Neurosci 34:418–432.
26. Ignashchenkova A, Dicke PW, Haarmeier T, Thier P (2004) Neuron-speciﬁc
contribution of the superior colliculus to overt and covert shifts of attention. Nat
Neurosci 7:56–64.
27. Mai JK, Assheuer J, Paxinos G (1997) Atlas of the Human Brain (Academic, San Diego).

28. Sereno AB, Briand KA, Amador SC, Szapiel SV (2006) Disruption of reﬂexive attention
and eye movements in an individual with a collicular lesion. J Clin Exp Neuropsychol
28:145–166.
29. Akiyama T, et al. (2007) Unilateral amygdala lesions hamper attentional orienting
triggered by gaze direction. Cereb Cortex 17:2593–2600.
30. Sander D, Grafman J, Zalla T (2003) The human amygdala: An evolved system for
relevance detection. Rev Neurosci 14:303–316.
31. Costafreda SG, Brammer MJ, David AS, Fu CHY (2008) Predictors of amygdala
activation during the processing of emotional stimuli: A meta-analysis of 385 PET and
fMRI studies. Brain Res Rev 58:57–70.
32. Sah P, Faber ESL, Lopez De Armentia M, Power J (2003) The amygdaloid complex:
Anatomy and physiology. Physiol Rev 83:803–834.
33. Davis M, Whalen PJ (2001) The amygdala: Vigilance and emotion. Mol Psychiatry 6:
13–34.
34. Lim MM, Young LJ (2006) Neuropeptidergic regulation of afﬁliative behavior andsocial bonding in animals. Horm Behav 50:506–517.
35. Di Simplicio M, Massey-Chase R, Cowen PJ, Harmer CJ (2009) Oxytocin enhances
processing of positive versus negative emotional information in healthy malevolunteers. J Psychopharmacol 23:241–248.
36. Unkelbach C, Guastella AJ, Forgas JP (2008) Oxytocin selectively facilitates recognitionof positive sex and relationship words. Psychol Sci 19:1092–1094.
37. Heinrichs M, Domes G (2008) Neuropeptides and social behaviour: Effects of oxytocin
and vasopressin in humans. Prog Brain Res 170:337–350.
38. Critchley HD, et al. (2000) The functional neuroanatomy of social behaviour: Changes
in cerebral blood ﬂow when people with autistic disorder process facial expressions.
Brain 123:2203–2212.
39. Ashwin C, Baron-Cohen S, Wheelwright S, O’Riordan M, Bullmore ET (2007)
Differential activation of the amygdala and the ‘social brain’ during fearful faceprocessing in Asperger Syndrome. Neuropsychologia 45:2–14.
40. Ashwin C, Chapman E, Colle L, Baron-Cohen S (2006) Impaired recognition ofnegative basic emotions in autism: A test of the amygdala theory. Soc Neurosci 1:
349–363.
41. Pelphrey KA, et al. (2002) Visual scanning of faces in autism. J Autism Dev Disord 32:
249–261.
42. Guastella AJ, et al. (2010) Intranasal oxytocin improves emotion recognition for youth
with autism spectrum disorders. Biol Psychiatry 67:692–694.
43. Andari E, et al. (2010) Promoting social behavior with oxytocin in high-functioning
autism spectrum disorders. Proc Natl Acad Sci USA 107:4389–4394.
44. Phan KL, Fitzgerald DA, Nathan PJ, Tancer ME (2006) Association between amygdala
hyperactivity to harsh faces and severity of social anxiety in generalized social phobia.
Biol Psychiatry 59:424–429.
45. Horley K, Williams LM, Gonsalvez C, Gordon E (2004) Face to face: Visual scanpath
evidence for abnormal processing of facial expressions in social phobia. Psychiatry Res
127:43–53.
46. Arrais KC, et al. (2009) Social anxiety disorder women easily recognize fearfull, sad andhappy faces: The inﬂuence of gender. J Psychiatr Res, 10.1016/j.jpsychires.2009.11.003.
47. Amin N, Foa EB, Coles ME (1998) Negative interpretation bias in social phobia. Behav
Res Ther 36:945–957.
48. Guastella AJ, Howard AL, Dadds MR, Mitchell P, Carson DS (2009) A randomized
controlled trial of intranasal oxytocin as an adjunct to exposure therapy for social
anxiety disorder. Psychoneuroendocrinology 34:917–923.
49. Hoffman KL, Gothard KM, Schmid MC, Logothetis NK (2007) Facial-expression andgaze-selective responses in the monkey amygdala. Curr Biol 17:766–772.
50. Heinrichs M, Gaab J (2007) Neuroendocrine mechanisms of stress and social
interaction: Implications for mental disorders. Curr Opin Psychiatry 20:158–162.
51. Born J, et al. (2002) Snifﬁng neuropeptides: A transnasal approach to the humanbrain. Nat Neurosci 5:514–516.
52. Tzourio-Mazoyer N, et al. (2002) Automated anatomical labeling of activations in
SPM using a macroscopic anatomical parcellation of the MNI MRI single-subject brain.
Neuroimage 15:273–289.

NEUROSCIENCEACKNOWLEDGMENTS. We thank G. Müller, K. Müller, and K. Wendt for help
with MR scanning. This study was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF, Network “Social Cognition”).

SEE COMMENTARY

activation clusters with high-resolution diagrams of the human amygdala as
depicted in an anatomical atlas (27). For illustration purposes, only voxels are
displayed as statistical maps thresholded at P < 0.01, uncorrected, which areoverlaid on the mean structural image from all participants. This mean image was generated from the high-resolution T1 images that were normalized using DARTEL ﬂow ﬁelds.

coordinate, see Results). These time series were then used as regressors insubject-speciﬁc design matrices, and simple contrast maps were calculated
reﬂecting a correlation of the seed region with the whole measured brain
volume. At the second level, a two-sample t test was carried out to determine brain regions showing an enhanced coupling with the left superior
colliculus in the oxytocin as compared with the placebo group.
Because we were primarily interested in modulatory effects of oxytocin on
amygdala activation, we used a small volume correction in predeﬁned bilateral anatomical amygdala regions of interest (52). Anatomical labels for
subregions within the amygdala were speciﬁed by comparing the location ofGamer et al.

PNAS | May 18, 2010 | vol. 107 | no. 20 | 9405