Title

Developmental changes in social attention andoxytocin levels in infants and children

Author(s)西里, 美菜保Citation

大阪大学, 2017, 博士論文

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https://doi.org/10.18910/67077

rights
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Osaka University Knowledge Archive : OUKA
https://ir.library.osaka-u.ac.jp/
Osaka University

Developmental changes in social attention and oxytocin levels in infants and children（ 乳幼児期における社会的注意とオキシトシン濃度の発達的変化 ）大阪大学大学院
大阪大学・金沢大学・浜松医科大学・千葉大学・福井大学
連合小児発達学研究科
小児発達学専攻

西里 美菜保

２０１７年６月 博士学位論文

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OPEN

Received: 26 October 2016
Accepted: 10 April 2017
Published: xx xx xxxx

Developmental changes in social
attention and oxytocin levels in
infants and children
Minaho Nishizato1, Takashi X. Fujisawa1,2, Hirotaka Kosaka1,2 & Akemi Tomoda1,2
Oxytocin (OT) signalling represents one of the most critical systems involved in human social behaviour.
Although several studies have examined the relationship between social functioning and peripheral OT
levels, the association between OT and the development of social attention has not been well studied.
Therefore, we investigated the developmental relationship between gaze fixation for social cues and
OT levels during young childhood. We examined visual attention using an eye tracking system in infants
and children (5–90 months of age) and measured the concentration of OT in saliva samples. We observeda negative association between age and both attention toward social cues and salivary OT levels, and apositive association between age and attention for non-social cues. We also observed that salivary OTlevels were modulated by polymorphisms in oxytocin receptor (OXTR) rs53576. Our results suggest that
there is an age-dependent association between visual attention for social cues and OT levels in infants
and children, and that the development of visual attention to the eyes as social cues is associated withboth OXTR polymorphisms and OT levels. Such findings indicate that OT and OXTR status may provide
insight into the atypical development of social attention in infants and young children.
Oxytocin (OT), a neuropeptide secreted from the posterior pituitary, has physiological functions in labour andlactation, and increasing evidence indicates that OT plays an important role in modulating social behaviour indiverse species1. In humans, much research has suggested that OT facilitates the ability to infer the mental state ofothers from viewing the eye region2, 3 and can even selectively enhance the memory encoding of faces4. OT also
modulates trust and generosity in interpersonal relationships5, 6. In fact, OT affects the activation of brain areas
responsible for emotion, mentalisation, and cognitive control, including the amygdala and prefrontal cortex5, 7.
In addition, research has suggested that oxytocin receptors (OXTRs) are expressed within specific brain areas
such as the prefrontal cortex, cingulate cortex, and amygdala in rodents and sheep8, as well as in humans9. These
areas are involved in social behaviour, including reproductive and maternal behaviours, affiliation and attachment, and reactivity to social stress in nonhuman mammals10. In humans, these brain areas are also referred toas “social-brain networks”11, 12. Thus, OT and OXTR, which comprise the oxytocinergic system, play crucial roles
in human social behaviour.
Genetic polymorphisms that modulate oxytocin neurotransmission have been linked to individual differences in social behavior13. Specifically, the OXTR gene is considered to play an important role in regulating social
behaviour by modulating the release of oxytocin14. A common, naturally-occurring single-nucleotide polymorphism (SNP)—OXTR rs53576—has been observed in an intron of the OXTR gene in humans.When presented
with social stimuli (emotional faces), adults with the AA genotype exhibit decreased amygdala activation andincreases in the functional correlation between the hypothalamus and amygdala compared with adults with the
AG or GG genotypes15. At the behavioural level, a significant correlation between affective mutuality in parent–child interactions and empathy for children was observed in children with the GG genotype, but not for children
with an A allele16. According to a recent meta-analysis, there are positive associations between the rs53576 polymorphism and general sociality: G allele homozygotes had higher general sociality than the A allele carriers17.
Collectively, the literature suggests that the OXTR rs53576 polymorphism is associated with systematic neuraland behavioural differences in social functioning.
1

Division of Developmental Higher Brain Functions, United Graduate School of Child Development, University of
Fukui, Fukui, 23-3 Matsuoka-Shimoaizuki, Eiheiji-cho, Fukui 910-1193, Japan. 2Research Center for Child Mental
Development, University of Fukui, 23-3 Matsuoka-Shimoaizuki, Eiheiji-cho, Fukui, 910-1193, Japan. Minaho
Nishizato and Takashi X. Fujisawa contributed equally to this work. Correspondence and requests for materials
should be addressed to A.T. (email: atomoda@u-fukui.ac.jp)
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Human Face(without mouth
motion)Human Face (withmouth motion)

People & Geometry

Biological Motion

Finger pointing%Eyes

Total (n = 149)

Boys (n = 76)

Girls (n = 73)

M

SD

M

SD

M

SD

t

Age

Correlation
OT levels

68.61

19.18

66.89

20.26

70.39

17.94

−1.11

−0.421***0.238**%Mouth

16.58

14.91

15.99

14.92

17.19

14.98

−0.49

0.531***−0.273**%Out of AOI

14.81

13.10

17.12

15.15

12.42

10.11

2.24*

0.011

−0.038

%Eyes

25.35

18.27

25.98

17.58

24.69

19.05

0.43

−0.198*0.243**%Mouth

58.25

21.85

57.83

21.52

58.68

22.18

−0.23

0.287***−0.260**%Out of AOI

16.40

12.91

16.18

12.60

16.63

13.31

−0.21

−0.205*0.097%People

52.12

15.37

48.86

16.07

55.51

13.92

−2.7**−0.417***−0.099%Geometry

27.16

15.99

29.90

16.78

24.32

14.71

2.15*

0.517***

0.056%Out of AOI

20.71

7.67

21.24

8.53

20.16

6.67

0.86

−0.244**

0.082%Upright

56.56

15.42

54.36

14.68

58.84

15.94

−1.79

0.096

−0.124%Inverted

34.26

14.54

35.35

13.66

33.12

15.42

0.93

−0.0060.143%Out of AOI

9.18

10.46

10.29

12.62

8.03

7.50

1.33

−0.133−0.017%Pointed

49.33

10.86

47.54

10.46

51.18

11.02

−2.07*−0.395**0.080%Non−pointed

9.49

7.41

10.12

7.64

8.82

7.15

1.07

0.526**−0.186*%Out of AOI

41.19

10.18

42.33

10.18

40.00

10.11

1.41

0.039

0.059

73.26

63.43

68.94

55.97

77.76

70.47

−0.85

−0.438***—

Oxytocin levels (pg/ml)

Table 1. Mean values and coefficients of salivary OT levels and gaze fixation parameters. *p < 0.05, ** p < 0.01,***p < 0.001. Abbreviations: AOI, area of interest; OT, oxytocin.

The duration of eye gazing is predictive of one’s ability to interpret the intentions of others and the meaning ofsocial situations18. Developmental research has also suggested that gaze fixation plays a key role in social development19, 20. Eye-tracking technology has several advantages for investigating visual attention to social cues intypical or atypical development during infancy and childhood21. Several studies have used eye tracking software
to compare atypical responses for social cues (e.g., human upper body, geometric patterns, or social images, etc.),such as those that occur in autism spectrum disorders (ASD), to those of individuals who have undergone typical development22, 23. This approach enables researchers to measure, with high precision and accuracy, at what a
participant is looking and for how long. Moreover, it offers an optimal balance between ecological validity andmethodological constraints24. Eye tracking is therefore a valuable method for the detection and characterisationof subtle variations in patterns of visual attention to social cues. Moreover, eye-tracking technology is applicable
to all populations from infants to adults, irrespective of their level of non-verbal and verbal ability24. Therefore,
the different aspects of visual attention for social cues can be investigated similarly across various participant
statuses, such as age, gender, and clinical condition.
The aim of the present study was to investigate the relationship between visual attention for social cues andsalivary OT levels in infants and children, as well as the potential modulation of these factors by the OXTR
rs53576 gene polymorphism. Although several studies have utilised eye-tracking to examine the relationshipbetween social functioning and peripheral OT levels, or that between social dysfunction and the pattern of visual
attention, few studies have investigated the associations between the two during typical development. Previously,
we revealed that aging in preschool children has a considerable effect on visual attention toward social cues25.
However, developmental changes in this process occurring during infancy and young childhood remain largely
unexplored, and the interaction between developmental changes in OT concentration and visual attention
remains unclear. Therefore, in the present study, we investigated the relationships among these factors by measuring patterns of visual attention for social cues and salivary OT levels using an eye-tracking system and an
enzyme-linked immunoabsorbant assay, respectively. Further, we identified OXTR polymorphisms using SNP
genotyping analysis. We hypothesized that low visual attention for social cues would be associated with low salivary OT levels and/or the OXTR “risk” allele, and that the associations between these factors would be modulated
by developmental changes.

Results

Developmental changes in visual attention for social cues. The mean percentage of time spent
fixated on each category of social cues is presented in Table 1, together with the standard deviation. We first
determined whether there were any sex differences in fixation duration using separate independent t-tests foreach variable. First, for “human face” stimuli with or without mouth motion, no significant sex differences wereobserved in the percentage of fixation time for either area of interest (AOI) [eye area without mouth motion:t (149) = −1.11, p = 0.26; with mouth motion: t (149) = 0.43; and the mouth area without mouth motion: t(149) = −0.49, p = 0.62; with mouth motion: t (149) = −0.23, p = 0.82]. In contrast, for the “people and geometry” stimuli, a significant sex difference was observed in the percentage of fixation time for both AOIs [people
stimuli: t (147) = −2.70, p = 0.008; and geometry stimuli: t (147) = 2.15, p = 0.03], indicating that young girls
were more attentive to people moving and less attentive to geometric shapes than young boys. For bodily motion
stimuli, no significant sex difference was observed [Upright: t (149) = −1.79, p = 0.08; Inverted: t (149) = 0.93,
p = 0.35.]. Finally, for “finger pointing” stimuli, a significant sex difference was observed in the percentage of fixation time spent on pointed-at objects [t (147) = −2.07, p = 0.04], indicating that young girls were more attentive
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Figure 1. Correlations between age and percentage of the total time that eyes were fixated on social cues.
(a–d). The vertical axis indicates fixation duration on each type of social stimulus as a percentage of total timemeasured. The horizontal axis indicates the age of the children in months. **p < 0.01; ***p < 0.001.

to pointed-at objects than young boys, although no sex difference in attention for non-pointed-at objects wasobserved between groups [t (147) = 1.07, p = 0.28.].
Next, we investigated the influence of age on fixation time for each category in order to address whether thepattern of attention to social cues is dependent on age. The correlation coefficients between age and each variableof fixation duration are presented in Table 1. Significant negative correlations were observed between age and fixation time for most AOI-1s (i.e., the higher social cues, which included the eye area of the “human face” category,people moving in the “people and geometry” category, and pointed-at objects in the “finger-pointing” category),
with the exception of the upright figure in the “biological motion” category (Fig. 1). In addition, significant positive correlations were observed between age and fixation time for most AOI-2s (i.e., the non-social or lower social
control cues, which included the mouth area in the “human face” category, geometric patterns in the “people andgeometry” category, and non-pointed-at objects in the “finger pointing” category), again with the exception of
the inverted figure in the “biological motion” category. These results suggest that visual attention for higher social
cues decreases with age, whereas attention for non-social or lower social cues increases with age. This may be
associated with increased behaviours related to exploration of the external environments and/or investigation of
the associations between social cues and other non-social objects (e.g., joint attention).

Developmental changes in salivary oxytocin levels. Mean values of salivary OT levels are also presented in Table 1. No significant sex difference was observed with regard to salivary OT levels [t (149) = 0.85,
p = 0.44]. We then investigated the potential influence of age on salivary OT levels (Table 1). A significant negative correlation was observed between salivary OT levels and age, suggesting that salivary OT levels decrease withage (Fig. 2).
In a subset of participants, we further examined whether OT levels are affected by age-associated fluctuationsin levels of total protein (TP). We observed a significant negative association between age and salivary OT levels
normalised by salivary TP levels, suggesting that salivary OT levels decrease with age regardless of TP levels(Supplementary Information).
Relationship between gaze fixation duration and salivary OT levels. Because we observed parallel
decreases in attention for social cues and salivary OT levels with age, we then examined the direct associations
among these variables. As shown in Table 1, most of the significant correlations between gaze fixation durationand salivary OT levels were noted in the “human face” category. Positive correlations were observed between
OT levels and the fixation time spent on the eye area of the face [without mouth motion: r = 0.238, p = 0.003;
with mouth motion: r = 0.243, p = 0.003], whereas negative correlations were observed between OT levels andScientific Reports | 7: 2540 | DOI:10.1038/s41598-017-02368-x

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Figure 2. Relationship between age and salivary oxytocin (OT) levels. The vertical axis indicates salivary OTlevels (pg/ml), whereas the horizontal axis indicates age. **p < 0.01.

the fixation time spent on the mouth area [without mouth motion: r = −0.273, p = 0.001; with mouth motion:r = −0.260, p = 0.001]. These results suggest that children with higher OT levels were more attentive to the eyeregion in human faces than children with lower OT levels. Next, because both of these factors were influenced
by age, we calculated partial correlation coefficients between salivary OT levels and the fixation times spent oneach AOI in the face, controlling for age. Results indicated a weak yet significant positive partial correlation witha small effect size26 between OT levels and fixation time on the eye area in the “human face with mouth motion”
category (r = 0.176, p = 0.03), indicating that children with higher OT levels were more attentive to the eye area ofthe face (with mouth motion) than children with lower OT levels, regardless of their age.
We then conducted a path analysis to examine whether OT levels mediate the relationship between age andvisual attention to the eyes (Fig. 3). As shown in Fig. 3a, we observed that age was a significant predictor of gaze
fixation on the eye area for infants and children in the present study (human face [without mouth motion])(β = −0.181, t = 2.24, p = 0.025). As shown in Fig. 3b, age was also a significant predictor of oxytocin levels(β = −0.44, t = 5.99, p < 0.001). Furthermore, when oxytocin levels and age were entered simultaneously as predictors of fixation on the eye area (human face [without mouth motion]), oxytocin levels remained a significant
predictor (β = 0.202, t = 2.29, p = 0.022), while age did not (β = 0.009, t = 1.03, p = 0.30). Thus, these findings
suggest that oxytocin levels mediate the relationship between age and visual attention to the eye area (Human face[without mouth motion]).

OXTR rs53576 polymorphism and salivary OT levels.

The distribution of genotypes for OXTR
(rs53576) in the participant group was as follows: 22 GG (14.8%), 65 AG (43.6%), and 62 AA (41.6%) genotypes.
Prior to statistical analysis, we confirmed that no significant deviation from the Hardy–Weinberg equilibrium wasobserved in participants [χ2(1) = 0.53, p = 0.47]. This distribution was similar to those observed in other studies
involving Asian participants27.
To examine the effect of the OXTR rs53576 genotype on salivary OT levels, a one-way analysis of variance(ANOVA) was performed using participants’ genotypes as a between-participants factor (Fig. 4). The analyses
revealed a significant main effect of genotype [F (2,146) = 4.461, p = 0.01]. Post hoc analysis using Shaffer’s modified sequentially rejective Bonferroni procedure revealed that individuals with the AA homozygote genotype(mean = 56.72, SD = 45.17) had lower salivary OT levels than individuals with a G allele (AG: mean = 80.54,
SD = 68.08; GG: mean = 98.37, SD = 77.86, respectively). This result suggests that salivary OT levels in young
children are modulated by the OXTR rs53576 polymorphism, with AA genotype carriers exhibiting significantly
lower salivary OT levels than G allele carriers.

Effects of age and OXTR polymorphisms on gaze fixation for social cues. Human cognitive development, including visual attention, appears to change from the sensorimotor period to the preoperational period,
which occurs at the age of approximately 24 months28, 29. Research has also indicated that the role of gaze fixation
may change significantly over the second year of life30. Therefore, the data were analysed to ascertain whether aninteraction effect between age and OXTR polymorphisms existed in relation to visual attention for social cues.
A two-way ANOVA was conducted, including age below 24 months and from 24 months and older, OXTR polymorphisms (AA genotype or G allele), and the fixation duration of each AOI in the face. This analysis indicatedthat only the fixation duration for the eye area of the face (without mouth motion) was associated with a significant interaction between age and OXTR genotype from 24 months onwards [F (1,145) = 5.53, p = 0.02]. No main
was observed for either genotype or age (Fig. 5).
There were no significant associations in any other AOIs of the face. Upon further examination of the interactions, post hoc analysis revealed a simple main effect of genotype, indicating that children over 24 months of age
with the AA genotype exhibited a significantly decreased fixation duration on the eye area compared to G allele

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Figure 3. The role of oxytocin in mediating the influence of infant age on gaze fixation on the eye area (Humanface [without mouth motion]). *p < 0.05; ***p < 0.001.

Figure 4. Relationship between OXTR polymorphisms and salivary oxytocin (OT) levels. The vertical axis
indicates salivary OT levels (pg/ml). The horizontal axis indicates the OXTR genotypes. *p < 0.05; **p < 0.01.
carriers [F (1,145) = 4.07, p = 0.04]. No simple main effect of genotype was observed in children under 24 monthsof age [F (1,145) = 1.75, p = 0.19].

Discussion

We investigated the developmental relationships between gaze fixation for social cues and OT levels in infants andyoung children between the ages of 6 and 90 months. Our analysis yielded four main findings. First, we observeda parallel decrease in both attention to social cues (e.g. eyes, people, or finger pointing) and salivary OT levels
with age during infancy and early childhood, as well as an increase in attention for non-social cues (e.g. geometry
or non-pointed-at objects). Second, we observed significant associations between attention to the eye and mouthareas of the face and salivary OT levels. Third, we also revealed that children with the OXTR AA homozygous
genotype exhibited lower salivary OT levels than those with G alleles. Finally, visual attention on the eye area wasassociated with both OXTR polymorphisms and age.
Several previous studies have suggested that OT exerts a positive effect on attention for social cues, including
the eye region1–3, 7. Further, impairments in social functioning, such as those associated with ASD, have been
related to dysfunction of the oxytocinergic system31–33. Previous eye-tracking studies have revealed that children
with atypical development (including those with ASD) exhibit lower or altered visual attention to social cues24.
Although we have examined such associations in our previous studies22, 34, 35, to the best of our knowledge, the

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Figure 5. Interaction between genotype and age in visual attention to the eyes as social cues. Solid line is the
OXTR rs53576 AA group (n = 62); dotted line is the OXTR G-carrier group (n = 87). *p < 0.05.

present study is the first to clarify the association between developmental changes in the pattern of visual attention to the eyes as social cues and OT levels during young childhood.
Our results offer new information, indicating that attention to most types of social cues in infants and young
children decrease with age, whereas attention to non-social cues increases with age. Additionally, the trajectories
of age-related changes in social attention may be dependent on the types of social cues used (e.g., face, people,
or finger pointing). Although a handful of studies regarding developmental changes in visual attention for social
cues have been published22, 34, 35, the decrease observed in the present study is partially consistent with the findings of a previous study, which reported that gaze fixation increases with age in infants35.
Our results also indicate that salivary OT levels decrease with age. Although only a handful of studies haveinvestigated the effects of age on the human oxytocin system36, age-related decreases in OT levels have been
observed in infant rhesus macaques37. Furthermore, one previous study revealed that mother-infant interactions
are positively associated with endogenous OT levels in human mothers and their children14. These findings suggest that the observed decrease in OT levels may reflect a reduction in direct interaction between children andtheir mothers, such as physical contact or being together, with aging (e.g., infants spend more time interactingwith their mothers than toddlers or younger children).
The most curious finding of the present study was the significant parallel decrease between salivary OT levels
and the duration of gaze fixation on the eye and mouth areas. When controlled for age, weak positive correlations
between OT levels and gaze fixation on the eye area (with mouth motion) were observed. Research on humancommunication has suggested that OT facilitates the ability to infer the mental state of others from the eye regionin adults2, 3, 32 and during parent-infant interactions14, 38. Therefore, we speculate that higher OT during young
childhood plays a significant role in executing efficient social interactions between the mother and child via eye
contact.
In the present study, we observed that children with the AA homozygous genotype had lower salivary OTlevels than those with G alleles. However, previous studies have suggested no association between peripheral OT
levels and OXTR rs53576 polymorphisms in either children or adults39, 40. The inconsistency between our findings
and those of previous studies may be attributable in part to differences in OT levels or OXTR genotype frequencies based on demographic characteristics of the population, such as age or ethnicity39, 40.
As previously reported17, 41, the OXTR rs53576 AA homozygous genotype, associated with decreased sociality,
was associated with lower OT levels in the present study. Further, OXTR rs53576 AA homozygotes demonstrated
lower fixation duration on the eye area compared to G-allele carriers among children aged 24 months and older.
Only one previous study has reported an association between visual attention to the eyes and a gene polymorphism related to oxytocin (CD38) in young children42. Together, these studies suggest that social eye cues can be
manipulated by genetic factors as well as endogenous levels of oxytocin. Although our findings are largely consistent with those of the previous study, the interaction was apparent only after 24 months of age in the current
study. One possible explanation for this is the establishment of multidimensional processes of social synchrony
with other social bonds, such as those with peers, after 24 months of age28, 43. With continued investigation, thisline of research may aid in the development of critical early screening tools, allowing for the identification of

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infants at high risk for developing social impairments based on OXTR polymorphisms and low OT levels in earlychildhood.
Several limitations of the present study should be noted and taken into consideration in future studies. First,
this study included a relatively small sample of participants and utilised a cross-sectional design that precluded
the identification of causal links between the development of social attention and OT systems. Longitudinal studies utilizing larger sample sizes are required in order to more fully elucidate the association between developmentof the OT system and visual attention for social cues. Second, although we used unextracted samples to measure
OT levels for two major reasons in the current study (see Methods), recent studies have reported inconsistentfindings regarding the relationship between extracted and unextracted samples for the measurement of peripheral OT levels44, 45. Further research is also required to more fully elucidate the neurobiological determinants ofsalivary OT levels. In addition, we were unable to examine the effect of TP levels on OT levels46 in all participants,
as the volume of salivary samples was insufficient in some participants. Although we observed an association
between OT levels and age regardless of TP levels in a subset of participants, future studies should verify this finding by measuring salivary OT levels for all samples obtained. Third, we did not exclude developmental disabilities
from our analyses or assess socioeconomic status and individual parenting style. Hence, we cannot conclude
whether our approach (i.e., the combination of eye-tracking using the Gazefinder, measurement of salivary OTlevels, and assessment of OXTR polymorphisms) is useful for early screening in infants. Fourth, the non-invasivenature of the Gazefinder, which provides new insight into social function, may be of particular advantage inthe examination of children, allowing clinicians to monitor patterns of gaze fixation when screening for ASD.
However, our findings regarding social functioning in the present study do not necessarily imply diagnostic
importance, as the data are descriptive of physical and functional condition. Finally, other polymorphisms or
gene-gene interactions were not taken into consideration47, 48. As recently suggested, ethnic differences in OXTR
genetic effects and certain forms of culture-specific relational norms may also be important27. Therefore, futurestudies should also include OXTR polymorphism data in the path analysis.
In conclusion, our results demonstrate that both attention for social cues (eyes, people, pointing area) andsalivary OT levels are negatively associated with age, whereas attention for non-social cues (mouth, geometry,non-pointed area) is positively associated with age. Furthermore, OT levels are positively associated with visual
attention for human faces, especially for the eye area. These results suggest that OT is involved in visual attention
for eyes but that such associations are largely dependent on age. In addition, OT levels are modulated by the
OXTR rs53576 polymorphism in children after 24 months of age, and the AA genotype is significantly associatedwith decreased fixation time on the eye area, relative to that in children with G alleles. These results suggest thatthe development of attention for eyes is modulated by the OXTR polymorphism, which exerts an effect on OT
levels. Thus, combining this experimental paradigm with neurophysiological indicators of brain activity (suchas imaging techniques) should prove fruitful in further elucidating mechanisms underlying visual attention forsocial cues.

Methods

Participants. One hundred and forty-nine infants and children (76 boys, 73 girls; mean age: 33.6 ± 24.3
months; age range: 5–90 months) and their mothers, who were recruited from the local community via advertisements, participated in the present study. The ethnicity of all participants was Japanese. We did not perform
psychodevelopmental evaluations of the participants. However, if the presence of developmental problems was
suspected in candidates for this study, we excluded them from our recruitment. No participants had any history of
any form of epilepsy or abuse, head injury, or foetal drug exposure that might have influenced brain development.
We did not measure socioeconomic status.
The study protocol was approved by the Ethics Committee of the University of Fukui (Assurance no.
20140142) and was conducted in accordance with the Declaration of Helsinki and the Ethical Guidelines forClinical Studies of the Ministry of Health, Labour, and Welfare of Japan. The parent(s) of all participants provided
written informed assent and consent for participation in this study.
Measurement of gaze patterns.

We measured each participant’s gaze pattern using Gazefinder (JVCKENWOOD Corporation, Kanagawa, Japan), an all-in-one eye-tracking system for responses to social cues byvisual stimuli25, 49. The Gazefinder used infrared light sources and cameras that were integrated into a 19-inch-thinfilm transistor monitor (1280 × 1024 pixels). Using corneal reflection techniques, the Gazefinder records the X
and Y coordinates of each child’s eye position at a frequency of 50 Hz (i.e., 3000 data collections/minute). Stimuli
presented by the Gazefinder consisted of short movies including four categories of social cues, which were (a)human faces (with or without mouth motion), (b) people and geometric patterns, (c) biological motion of ahuman, and (d) objects with or without finger pointing. Additionally, the category (a) of human faces was divided
into two subcategories. One subcategory is included stimuli without mouth motion, such as staring with silence,
while the other included stimuli with mouth motion, such as talking (e.g., “Hello” or “What’s your name?”).
Moreover, two areas-of-interest (AOI-1 and AOI-2) were set within each stimulus. AOI-1 was a target area
considered to represent higher social cues [“eyes” in (a), “people” in (b), “upright figure” in (c), and “object withpointing” in (d)]. AOI-2 was a control area considered to represent lower or non-social cues [“mouth” in (a),“geometry” in (b), “inverted figure” in (c), and “objects without pointing” in (d)] (For more details, refer to previous studies25, 49).

Procedure and stimuli.

The experiments were conducted at a research laboratory located in the ResearchCenter for Child Mental Development at the University of Fukui, Japan. Children were seated (on a parent’s lap
when younger than 24 months old, and mothers were instructed not to assist their children) 40 cm in front of the

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eye-tracking monitor. To obtain calibration information, the participants were initially asked to look at imagesof an animated animal that appeared in one of five locations on the screen. If the calibration quality was poor for
any of these points, the calibration process was repeated. Before the task, children were instructed that picturesof faces, people, and objects were to be shown on the computer screen and that they should look at them withoutlooking away for as long as possible. Stimulus movies were displayed in a definitive order49. Before each trial, an
attention-getting animation with a voice saying “Hey! Look!” was presented in the centre of the screen to reorient
the child’s attention to the stimuli.

®

Measurement of salivary OT levels. Saliva samples were collected using Salivettes (Sarstedt,
Rommelsdorft, Germany) after the children had viewed the stimulus movies. Parents were asked to put a roll ofcotton in their child’s mouth and instruct their child to chew for 1 minute until it was saturated with saliva. Two
cotton samples were collected by repeating this process. Saliva samples were frozen and stored at −80 °C. Before
the assay, saliva samples were lyophilized overnight and kept at −20 °C to concentrate them two to four times. The
dry samples were reconstituted in the assay buffer immediately before analysis using a commercial OT enzyme
immunoassay kit (Enzo Life Sciences, Inc., NY, USA). These protocols were consistent with those of an earlier
study on adults50–52, as well as with those of a study conducted with 3-year-old children14 and of our previous
study25.
In the current study, we used unextracted salivary samples for the following reasons. First, as analysis of
unextracted salivary samples revealed a more than 80% recovery rate as well as acceptable matrix interference,
we considered extraction unnecessary. Second, previous reports have suggested that the extraction process may
remove the majority of peripheral oxytocin, including oxytocin that is bound to other molecules in the blood,
such as albumin53. The kit utilised in the present study is exclusive to OT, eliminating the influence of other peptides such as arginine, vasopressin, and somatostatin. Each sample was examined in duplicate, and concentrations
were calculated using the SpectraMax (Molecular Device, Sunnyvale, California) micro plate reader, according
to relevant standard curves. Average intra-and inter-assay coefficients of variation (CV) were 6.5% and 9.4%,
respectively.
We also measured salivary protein levels using a commercial protein assay kit (Thermo Fisher ScientificInc., Waltham, MA, USA) to investigate the effect of TP on age-associated changes in OT levels (Supplementary
Information). However, this analysis was performed only for a subset of participants (n = 54), as the volume ofsaliva samples was insufficient for some infants and children. We also measured salivary blood contamination levels54 using a commercial EIA kit (Salimetrics Inc., State College, PA, USA) in a subset of participants. The resultsof this analysis revealed that the level of blood contamination was within the acceptable range for the samples(M = 0.43 ± 0.3 mg/dl).

®

Genotyping. Genomic DNA was extracted from the buccal mucosa cells using a standard phenol-chloroform

method using the QIAamp DNA Micro Kit (QIAGEN, Tokyo, Japan). We targeted the OXTR SNP (rs53576),
which was selected due to its significant association with “sociality” in human behaviour in a previous
meta-analysis17. SNPs were genotyped using TaqMan genotyping assays (Applied Biosystems, Foster City, CA,
USA) and the standard protocols provided by the manufacturer. All samples were genotyped via real-time polymerase chain reaction (PCR) analysis using the StepOnePlus System (Applied Biosystems, Foster City, CA,
USA). Reactions were performed in a 10 μL volume, containing 9 ng genomic DNA, 0.25 μL of Tris-EDTA buffer,
0.25 μL of each TaqMan probe, and 5 μL TaqMan PCR Master Mix. The PCR cycling conditions consisted of one
10-minute cycle at 95 °C, followed by 60 cycles at 95 °C for 30 seconds and 60 °C for 30 seconds. In each amplification, 4.5 μL HPLC water plus Master Mix was used as a negative PCR control. Genotype discrimination was thenconducted using StepOnePlus System software (version 3.0.1.).

Statistical analysis. We computed the percentage of time spent with gaze fixated on the two AOIs, as wellas on other areas on the screen, and analysed them as dependent variables. First, we calculated the correlation
coefficients to examine the associations between the percentage of fixation time for each AOI, OT levels, and age.
Next, we conducted an analysis of variance (ANOVA) to compare OT levels among the rs53576 polymorphisms.
Finally, an ANOVA was used to assess the effects of both genotype and age on social attention. The significancelevel was set to p < 0.05. Statistical analysis was conducted using R software (version 3.2.0. for Windows, R), and
ANOVAs were executed using “anovakun” in R software (version 4.8.0.)55.

References

1. Insel, T. R. The challenge of translation in social neuroscience: a review of oxytocin, vasopressin, and affiliative behavior. Neuron 65,
768–779, doi:10.1016/j.neuron.2010.03.005 (2010).
2. Domes, G., Heinrichs, M., Michel, A., Berger, C. & Herpertz, S. C. Oxytocin improves “mind-reading” in humans. Biol Psychiatry61, 731–733, doi:10.1016/j.biopsych.2006.07.015 (2007).
3. Guastella, A. J., Mitchell, P. B. & Dadds, M. R. Oxytocin increases gaze to the eye region of human faces. Biol Psychiatry 63, 3–5,
doi:10.1016/j.biopsych.2007.06.026 (2008).
4. Rimmele, U., Hediger, K., Heinrichs, M. & Klaver, P. Oxytocin makes a face in memory familiar. J Neurosci 29, 38–42, doi:10.1523/
JNEUROSCI.4260-08.2009 (2009).
5. Baumgartner, T., Heinrichs, M., Vonlanthen, A., Fischbacher, U. & Fehr, E. Oxytocin shapes the neural circuitry of trust and trust
adaptation in humans. Neuron 58, 639–650, doi:10.1016/j.neuron.2008.04.009 (2008).
6. Zak, P. J., Stanton, A. A. & Ahmadi, S. Oxytocin increases generosity in humans. PLoS One 2, e1128, doi:10.1371/journal.
pone.0001128 (2007).
7. Domes, G. et al. Oxytocin attenuates amygdala responses to emotional faces regardless of valence. Biol Psychiatry 62, 1187–1190,
doi:10.1016/j.biopsych.2007.03.025 (2007).
8. Skuse, D. H. & Gallagher, L. Dopaminergic-neuropeptide interactions in the social brain. Trends Cogn Sci 13, 27–35, doi:10.1016/j.
tics.2008.09.007 (2009).

Scientific Reports | 7: 2540 | DOI:10.1038/s41598-017-02368-x

8

www.nature.com/scientificreports/
9. Boccia, M. L., Petrusz, P., Suzuki, K., Marson, L. & Pedersen, C. A. Immunohistochemical localization of oxytocin receptors inhuman brain. Neuroscience 253, 155–164, doi:10.1016/j.neuroscience.2013.08.048 (2013).
10. Carter, C. S. Neuroendocrine Perspectives on Social Attachment and Love. Psychoneuroendocrinology 23, 779–818, doi:10.1016/
s0306-4530(98)00055-9 (1998).
11. Adolphs, R. The social brain: neural basis of social knowledge. Annu Rev Psychol 60, 693–716, doi:10.1146/annurev.
psych.60.110707.163514 (2009).
12. Feldman, R. The adaptive human parental brain: implications for children’s social development. Trends Neurosci 38, 387–399,
doi:10.1016/j.tins.2015.04.004 (2015).
13. Meyer-Lindenberg, A., Domes, G., Kirsch, P. & Heinrichs, M. Oxytocin and vasopressin in the human brain: social neuropeptides
for translational medicine. Nat Rev Neurosci 12, 524–538, doi:10.1038/nrn3044 (2011).
14. Feldman, R., Gordon, I., Influs, M., Gutbir, T. & Ebstein, R. P. Parental oxytocin and early caregiving jointly shape children’s oxytocin
response and social reciprocity. Neuropsychopharmacology 38, 1154–1162, doi:10.1038/npp.2013.22 (2013).
15. Tost, H. et al. A common allele in the oxytocin receptor gene (OXTR) impacts prosocial temperament and human hypothalamiclimbic structure and function. Proceedings of the National Academy of Sciences of the United States of America 107, 13936–13941,
doi:10.1073/pnas.1003296107 (2010).
16. McDonald, N. M., Baker, J. K. & Messinger, D. S. Oxytocin and Parent-Child Interaction in the Development of Empathy AmongChildren at Risk for Autism. Developmental Psychology 52, 735–745, doi:10.1037/dev0000104 (2016).
17. Li, J. et al. Association of Oxytocin Receptor Gene (OXTR) rs53576 Polymorphism with Sociality: A Meta-Analysis. PLoS One 10,
e0131820, doi:10.1371/journal.pone.0131820 (2015).
18. Klin, A., Jones, W., Schultz, R., Volkmar, F. & Cohen, D. Visual fixation patterns during viewing of naturalistic social situations aspredictors of social competence in individuals with autism. Archives of General Psychiatry 59, 809–816, doi:10.1001/
archpsyc.59.9.809 (2002).
19. Carpendale, J. I. M. & Lewis, C. Constructing an understanding of mind: The development of children’s social understanding withinsocial interaction. Behav. Brain Sci. 27, 79-+ (2004).
20. Jenkins, J. M. & Astington, J. W. Theory of mind and social behavior: Causal models tested in a longitudinal study. Merrill-Palmer
Q.-J. Dev. Psychol. 46, 203–220 (2000).
21. Karatekin, C. Eye tracking studies of normative and atypical development. Developmental Review 27, 283–348, doi:10.1016/j.
dr.2007.06.006 (2007).
22. Jones, W. & Klin, A. Attention to eyes is present but in decline in 2-6-month-old infants later diagnosed with autism. Nature 504,
427–431, doi:10.1038/nature12715 (2013).
23. Pierce, K., Conant, D., Hazin, R., Stoner, R. & Desmond, J. Preference for Geometric Patterns Early in Life as a Risk Factor for
Autism. Archives of General Psychiatry 68, 101–109, doi:10.1001/archgenpsychiatry.2010.113 (2011).
24. Guillon, Q., Hadjikhani, N., Baduel, S. & Roge, B. Visual social attention in autism spectrum disorder: insights from eye tracking
studies. Neurosci Biobehav Rev 42, 279–297, doi:10.1016/j.neubiorev.2014.03.013 (2014).
25. Fujisawa, T. X., Tanaka, S., Saito, D. N., Kosaka, H. & Tomoda, A. Visual attention for social information and salivary oxytocin levels
in preschool children with autism spectrum disorders: an eye-tracking study. Front Neurosci 8, 295, doi:10.3389/fnins.2014.00295(2014).
26. Cohen, J. Statistical power analysis for the behavioral sciences. (L. Erlbaum Associates, 1988).
27. Kim, H. S. et al. Culture, distress, and oxytocin receptor polymorphism (OXTR) interact to influence emotional support seeking.
Proc Natl Acad Sci USA 107, 15717–15721, doi:10.1073/pnas.1010830107 (2010).
28. Jean Piaget, B. I. The Psychology of the Child. (Basic Books 1969).
29. Thorgrimsson, G. B., Fawcett, C. & Liszkowski, U. 1- and 2-year-olds’ expectations about third-party communicative actions. Infant
Behav Dev 39, 53–66, doi:10.1016/j.infbeh.2015.02.002 (2015).
30. Moore, C. & Povinelli, D. J. Differences in How 12- and 24-Month-Olds Interpret the Gaze of Adults. Infancy 11, 215–231,
doi:10.1080/15250000701310348 (2007).
31. Andari, E. et al. Promoting social behavior with oxytocin in high-functioning autism spectrum disorders. Proc Natl Acad Sci USA
107, 4389–4394, doi:10.1073/pnas.0910249107 (2010).
32. Guastella, A. J. et al. Intranasal oxytocin improves emotion recognition for youth with autism spectrum disorders. Biol Psychiatry67, 692–694, doi:10.1016/j.biopsych.2009.09.020 (2010).
33. Kosaka, H. et al. Oxytocin efficacy is modulated by dosage and oxytocin receptor genotype in young adults with high-functioning
autism: a 24-week randomized clinical trial. Transl Psychiatry 6, e872, doi:10.1038/tp.2016.152 (2016).
34. Dupierrix, E. et al. Preference for human eyes in human infants. J Exp Child Psychol 123, 138–146, doi:10.1016/j.jecp.2013.12.010(2014).
35. Xiao, N. G. et al. Eye tracking reveals a crucial role for facial motion in recognition of faces by infants. Dev Psychol 51, 744–757,
doi:10.1037/dev0000019 (2015).
36. Huffmeijer, R., van Ijzendoorn, M. H. & Bakermans-Kranenburg, M. J. Ageing and oxytocin: a call for extending human oxytocin
research to ageing populations–a mini-review. Gerontology 59, 32–39, doi:10.1159/000341333 (2013).
37. Parker, K. J., Hoffman, C. L., Hyde, S. A., Cummings, C. S. & Maestripieri, D. Effects of age on cerebrospinal fluid oxytocin levels infree-ranging adult female and infant rhesus macaques. Behav Neurosci 124, 428–433, doi:10.1037/a0019576 (2010).
38. Feldman, R. et al. Sensitive parenting is associated with plasma oxytocin and polymorphisms in the OXTR and CD38 genes. Biol
Psychiatry 72, 175–181, doi:10.1016/j.biopsych.2011.12.025 (2012).
39. Chang, W. H. et al. Oxytocin receptor gene rs53576 polymorphism modulates oxytocin-dopamine interaction and neuroticism
traits–a SPECT study. Psychoneuroendocrinology 47, 212–220, doi:10.1016/j.psyneuen.2014.05.020 (2014).
40. Parker, K. J. et al. Plasma oxytocin concentrations and OXTR polymorphisms predict social impairments in children with andwithout autism spectrum disorder. Proceedings of the National Academy of Sciences of the United States of America 111, 12258–12263,
doi:10.1073/pnas.1402236111 (2014).
41. Clark, C. L. et al. Neonatal CSF oxytocin levels are associated with parent report of infant soothability and sociability.
Psychoneuroendocrinology 38, 1208–1212, doi:10.1016/j.psyneuen.2012.10.017 (2013).
42. Krol, K. M., Monakhov, M., Lai, P. S., Ebstein, R. P. & Grossmann, T. Genetic variation in CD38 and breastfeeding experienceinteract to impact infants’ attention to social eye cues. Proc Natl Acad Sci USA 112, E5434–5442, doi:10.1073/pnas.1506352112(2015).
43. Ulmer-Yaniv, A. et al. Affiliation, reward, and immune biomarkers coalesce to support social synchrony during periods of bondformation in humans. Brain, behavior, and immunity 56, 130–139, doi:10.1016/j.bbi.2016.02.017 (2016).
44. Robinson, K. J., Hazon, N., Lonergan, M. & Pomeroy, P. P. Validation of an enzyme-linked immunoassay (ELISA) for plasma
oxytocin in a novel mammal species reveals potential errors induced by sampling procedure. Journal of neuroscience methods 226,
73–79, doi:10.1016/j.jneumeth.2014.01.019 (2014).
45. Szeto, A. et al. Evaluation of enzyme immunoassay and radioimmunoassay methods for the measurement of plasma oxytocin.
Psychosomatic medicine 73, 393–400, doi:10.1097/PSY.0b013e31821df0c2 (2011).

Scientific Reports | 7: 2540 | DOI:10.1038/s41598-017-02368-x

9www.nature.com/scientificreports/
46. Feldman, R., Gordon, I. & Zagoory-Sharon, O. The cross-generation transmission of oxytocin in humans. Horm Behav 58, 669–676,
doi:10.1016/j.yhbeh.2010.06.005 (2010).
47. Aspe-Sanchez, M., Moreno, M., Rivera, M. I., Rossi, A. & Ewer, J. Oxytocin and Vasopressin Receptor Gene Polymorphisms: Role inSocial and Psychiatric Traits. Front Neurosci 9, 510, doi:10.3389/fnins.2015.00510 (2015).
48. Pappa, I., Mileva-Seitz, V. R., Bakermans-Kranenburg, M. J., Tiemeier, H. & van, I. M. H. The magnificent seven: A quantitative
review of dopamine receptor d4 and its association with child behavior. Neurosci Biobehav Rev 57, 175–186, doi:10.1016/j.
neubiorev.2015.08.009 (2015).
49. Fujioka, T. et al. Gazefinder as a clinical supplementary tool for discriminating between autism spectrum disorder and typicaldevelopment in male adolescents and adults. Mol Autism 7, 19, doi:10.1186/s13229-016-0083-y (2016).
50. Carter, C. S. et al. Oxytocin: behavioral associations and potential as a salivary biomarker. Ann N Y Acad Sci 1098, 312–322,
doi:10.1196/annals.1384.006 (2007).
51. Daughters, K. et al. Salivary Oxytocin Concentrations in Males following Intranasal Administration of Oxytocin: A Double-Blind,
Cross-Over Study. PLoS One 10, e0145104, doi:10.1371/journal.pone.0145104 (2015).
52. Lebowitz, E. R. et al. Salivary oxytocin in clinically anxious youth: Associations with separation anxiety and family accommodation.
Psychoneuroendocrinology 65, 35–43, doi:10.1016/j.psyneuen.2015.12.007 (2016).
53. Martin, W. & Carter, C. In 10th World Congress of Neurohypophyseal Hormones Abstracts, Bristol, UK.
54. Behr, G. A. et al. A statistical method to calculate blood contamination in the measurement of salivary hormones in healthy women.
Clin Biochem, doi:10.1016/j.clinbiochem.2016.12.005 (2016).
55. Anovakun (version 4.8.0), http://riseki.php.xdomain.jp/index.php/ (2016).

Acknowledgements

The authors wish to thank Shiho Tanaka and Madoka Umemoto for technical assistance with the experiments, andall of the research participants. This work was supported by a Grants-in-Aid for Scientific Research (B), Scientific
Research (C), and Challenging Exploratory Research from the Ministry of Education, Culture, Sports, Science,
and Technology (MEXT) of Japan (KAKENHI: grant numbers 15H03106 and 15K12720 to Akemi Tomoda, grant
number 15K01753 to Takashi X, Fujisawa); a Grant-in-Aid for “Creating a Safe and Secure Living Environment
in the Changing Public and Private Spheres” from the Japan Science and Technology Corporation (JST)/ResearchInstitute of Science and Technology for Society (RISTEX); and a research grant from the Danone Institute ofJapan Foundation (DIJF) to Akemi Tomoda. Part of this research was the result of the project “Research andDevelopment of Supportive Device Technology for Medicine Using ICT” from AMED (to Hirotaka Kosaka). The
funders had no role in the study design, data collection and analysis, decision to publish, or preparation of themanuscript.

Author Contributions

A.T. has full access to all the data in the study and takes full responsibility for the integrity of the data and theaccuracy of the data analysis. M.N. and T.X.F. performed the research, and/or conducted the statistical analysis.
M.N., T.X.F., H.K., and A.T. interpreted the data and took part in writing the manuscript. All authors haveapproved the final article.

Additional Information

Supplementary information accompanies this paper at doi:10.1038/s41598-017-02368-x
Competing Interests: The authors declare that they have no competing interests.
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