Oxytocin modulates fMRI responses to facial expression in ...administration in monkeys (20–25). Consistent with the human literature, these studies have found that intranasal administra-tion

Oxytocin modulates fMRI responses to facialexpression in macaquesNing Liua,1, Fadila Hadj-Bouzianea,2, Katherine B. Jonesa,3, Janita N. Turchib, Bruno B. Averbeckc,and Leslie G. Ungerleidera,1

aSection on Neurocircuitry, Laboratory of Brain and Cognition, NIMH, NIH, Bethesda, MD 20892; bSection on Cognitive Neuroscience, Laboratory ofNeuropsychology, NIMH, NIH, Bethesda, MD 20892; and cUnit on Learning and Decision Making, Laboratory of Neuropsychology, NIMH, NIH, Bethesda,MD 20892

Contributed by Leslie G. Ungerleider, April 29, 2015 (sent for review December 19, 2014; reviewed by Mark G. Baxter)

Increasing evidence has shown that oxytocin (OT), a mammalianhormone, modifies the way social stimuli are perceived and theway they affect behavior. Thus, OT may serve as a treatment forpsychiatric disorders, many of which are characterized by dysfunc-tional social behavior. To explore the neural mechanisms mediatingthe effects of OT in macaquemonkeys, we investigated whether OTwould modulate functional magnetic resonance imaging (fMRI)responses in face-responsive regions (faces vs. blank screen) evokedby the perception of various facial expressions (neutral, fearful,aggressive, and appeasing). In the placebo condition, we foundsignificantly increased activation for emotional (mainly fearful andappeasing) faces compared with neutral faces across the face-responsive regions. OT selectively, and differentially, altered fMRIresponses to emotional expressions, significantly reducing re-sponses to both fearful and aggressive faces in face-responsiveregions while leaving responses to appeasing as well as neutralfaces unchanged. We also found that OT administration selectivelyreduced functional coupling between the amygdala and areas in theoccipital and inferior temporal cortex during the viewing of fearfuland aggressive faces, but not during the viewing of neutral orappeasing faces. Taken together, our results indicate homologiesbetween monkeys and humans in the neural circuits mediating theeffects of OT. Thus, the monkey may be an ideal animal model toexplore the development of OT-based pharmacological strategiesfor treating patients with dysfunctional social behavior.

oxytocin | neuroimaging | nonhuman primate | social stimuli

In the last decade, oxytocin (OT), a mammalian hormone, hasbecome one of the most studied peptides of the neuroendo-

crine system. In humans, accumulating evidence has demon-strated that OT affects a wide range of social behavior andcognition, including perception, recognition and memory of so-cial stimuli (1–5), socially reinforced learning (6), and morecomplex sociocognitive behaviors [e.g., trust (7, 8), cooperation(9), generosity (10), and empathy (6, but see ref. 11)]. Therefore,it has been proposed that OT may serve as a treatment forvarious disorders with dysfunctional social behavior, such asautism spectrum disorders, antisocial personality disorder, andschizophrenia (for review, see ref. 12). A recent study found thatOT enhances brain activity for socially meaningful stimuli butattenuates activity for nonsocially meaningful stimuli in childrenwith autism spectrum disorders (13). Although these studiessuggest very promising prospects of OT for clinical use, theneural mechanisms underlying OT’s modulatory effects remainelusive. To understand these mechanisms, it is important to in-vestigate the effect of OT on brain activity, especially in regionsinvolved in social behavior and cognition.Functional magnetic resonance imaging (fMRI) has been the

major approach to investigating altered brain activation patternsin response to OT in humans. OT may affect the perception ofsocial stimuli, and thus mediate subsequent social informationprocessing (e.g., learning and memory, etc.) (14). Many fMRIstudies have examined the effects of OT on brain activity during

the perception of social stimuli to probe the brain regions thatunderlie OT’s modulatory effects. Emotional stimuli, which arecrucial for social communication and interaction, have beenmainly used. For example, Kirsch et al. showed that OT reducesactivation in response to fear-inducing stimuli in the amygdala, akey brain region involved in emotional regulation (15). Sub-sequently, a series of studies examined the effects of OT onresponses to facial expressions (3, 16), to conditioned facialexpressions (17), and to threatening scenes (18). These studiesshowed that activity evoked by emotional stimuli, especiallynegative stimuli (e.g., fearful faces, but see refs. 3 and 16 forhappy faces), is systematically altered within an interconnectednetwork of brain regions after OT administration.Because of the limitation of experimental approaches with

human subjects, animal models are essential not only for in-vestigating the neural mechanisms underlying the effects of OTbut also for exploring OT-based therapeutic strategies for in-dividuals with dysfunctional social behavior. Given the simi-larities between monkeys and humans in the neural circuitryunderlying social cognition (19), the rhesus macaque could be anideal animal model to examine the effects of OT. To date, only afew studies have investigated the behavioral consequences of OTadministration in monkeys (20–25). Consistent with the humanliterature, these studies have found that intranasal administra-tion of OT affects social behavior and cognition in monkeys,

Significance

Oxytocin (OT), a mammalian hormone, may serve as a treat-ment for psychiatric disorders because of its beneficial effecton social behavior. Here, we found that in monkeys, OT se-lectively altered brain activity within multiple neural systems(visual perception, emotion, attention, and higher cognitionfunction) and functional coupling between the amygdala andareas in the ventral visual pathway evoked by negative emo-tional expressions. Our findings provide key information forunderstanding the behavioral consequences of OT adminis-tration and indicate homologies between monkeys and hu-mans in the neural circuits mediating the effects of OT. Thus,the monkey may be an ideal animal model to explore the de-velopment of OT-based pharmacologic strategies for treatingpatients with dysfunctional social behavior.

Author contributions: N.L., F.H.-B., B.B.A., and L.G.U. designed research; N.L., K.B.J., andJ.N.T. performed research; N.L. and B.B.A. analyzed data; and N.L., F.H.-B., and L.G.U.wrote the paper.

Reviewers included: M.G.B., Mount Sinai School of Medicine.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.1To whom correspondence may be addressed. Email: [email protected] or [email protected].

2Present address: INSERM, U1028, CNRS UMR5292, Lyon Neuroscience Research Center,ImpAct Team - University UCBL Lyon 1, Lyon F-69000, France.

3Present address: Department of Psychology, Michigan State University, East Lansing,MI 48824.

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including vicarious as well as self-reinforcement (20), socialvigilance (22), socially reinforced learning (26), and attention tofacial features and expressions (21, 24). However, how OT exertsits effects on brain activity in monkeys remains unclear. To ex-plore the neural mechanisms mediating the effects of OT inmacaque monkeys, in the present study, we investigated whetherOT would modulate fMRI responses evoked by the perceptionof facial expressions, an effect mainly studied in humans thus far.We scanned monkeys while they viewed images of monkey

faces with four different expressions: neutral, fearful, aggressive,and appeasing. Scanning was conducting under two differentconditions: placebo control (saline) and intranasal OT. Wepredicted that in the placebo condition, emotional faces (espe-cially fearful) would evoke enhanced activation compared withneutral faces, that is, they would show a valence effect; that, as inhumans, OT administration would reduce this valence effect inmonkeys; and that OT administration would alter functionalcoupling among those brain regions showing a valence effect.

ResultsResponses to Neutral and Emotional Faces. Using the contrast ofneutral faces versus a blank screen in the localizer experiment,we found that face-responsive voxels were widely distributedbilaterally within the occipital cortex (V1, V2, V3, V4), withinthe lateral intraparietal sulcus (LIP), across the anterior portion(area TE) and the posterior portion (area TEO) of the inferiortemporal cortex, within and along the superior temporal sulcus (sts),within the dorsal portion of the lateral and basal nuclei of theamygdala, and within the prefrontal cortex (PFC), including thefrontal eye field (FEF), dorsolateral prefrontal cortex (DLPFC),ventrolateral prefrontal cortex (VLPFC), and orbitofrontal cortex(OFC) in all three subjects (Fig. 1A). Using the contrast ofemotional faces versus neutral faces, we found a similar, al-though spatially more limited, activation map (Fig. 1B).We performed an ANOVA with two within-subject factors

[region of interest (ROI) and expression] and one between-subject factor (treatment: placebo vs. OT). We found significantmain effects for ROI [F (11, 1,914) = 2,580.668; P < 0.001] andexpression [F (3, 522) = 44.010; P < 0.001], but not for treatment[F (1, 174) = 1.867; P = 0.174]. There were significant in-teractions between ROI and expression [F (33, 5,742) = 14.494;P < 0.001], ROI and treatment [F (11, 1,914) = 3.050; P < 0.001],and expression and treatment [F (3, 522) = 3.897; P = 0.009].The interaction among ROI, expression, and treatment was not

significant [F (33, 5,742) = 0.997; P = 0.473]. These findingsindicate that OT differentially altered fMRI responses to facialexpressions, and the effect was similar across face-responsiveROIs. To provide a complete picture of the effects of OT onresponses to neutral and emotional faces, we present the resultsfrom each ROI in detail by conducting post hoc analyses andtests for interactions, aware that the three-way interaction amongROI, expression, and treatment was not significant.

Placebo Condition. We evaluated responses to various facial ex-pressions by contrasting each category of emotional faces (fearful,aggressive, and appeasing) with neutral faces. This analysisshowed, relative to neutral faces, in all face-responsive ROIs,enhanced responses to fearful faces (P < 0.001) and appeasingfaces (V1: P = 0.037; V4: P = 0.026; TEO: P = 0.004; TE: P <0.001; LIP: P < 0.001; FEF: P < 0.001; DLPFC: P < 0.001;VLPFC: P < 0.001; amygdala: P = 0.010) except V2 (P = 0.110),V3 (P = 1.000), and OFC (P = 0.842). Responses to aggressivefaces did not significantly differ from those to neutral faces in thedefined ROIs. The group-averaged response profiles for eachROI in the placebo condition are illustrated in Figs. 2 and 3.

OT Condition.OT does not alter the responses to neutral faces. We first investigatedthe effect of OT on the fMRI signal evoked by neutral faces. Wefound no difference in the response to neutral faces between theOT and placebo conditions in any of the face-responsive ROIs,indicating that OT administration did not affect neutral faceprocessing (Figs. 2 and 3).OT modulates the valence effect.After OT administration, enhancedresponses to fearful faces relative to neutral faces observed in theplacebo condition were no longer present in V1 (P = 0.828), theamygdala (P = 1.000), or OFC (P = 0.275). Although enhancedresponses to fearful faces were still present in the other face-responsive ROIs after OT administration, significant or nearlysignificant interactions between treatment and valence [(fearfulvs. neutral in the placebo condition) vs. (fearful vs. neutral in theOT condition)] were found in all these ROIs (V2: P = 0.007; V4:P = 0.012; TEO: P = 0.007; TE: P = 0.004; LIP: P = 0.052; FEF:P = 0.037; DLPFC: P = 0.016; VLPFC: P = 0.003) except V3(P = 0.523). These interactions indicate that OT reduced theenhanced response to fearful relative to neutral faces (i.e., re-duced the valence effect for fearful faces). Because OT did notalter the response to neutral faces, OT administration mainly

Fig. 1. fMRI response to neutral faces comparedwith a blank screen in the localizer experiment andresponses to emotional faces compared with neutralfaces in the placebo condition. Face-responsive[neutral faces > blank screen, (A)] and valence effect[fearful/aggressive/appeasing faces > neutral faces(B)] activation maps are shown on lateral views ofthe right hemisphere of the inflated cortex (Top)and on coronal slices through the amygdala (Bot-tom) of monkey P. The locations of the coronal slicesare marked by the black line on the lateral views.The color bars show the statistical values of thecontrast between either neutral faces and a blankscreen or between emotional and neutral faces. as,arcuate sulcus; ios, inferior occipital sulcus; ls, lateralsulcus; lus, lunate sulcus; pmts, posterior middletemporal sulcus; sts, superior temporal sulcus.

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caused a reduction in the response evoked by fearful faces(fearful faces in the placebo condition vs. in the OT condition:V1: P < 0.001; V2: P = 0.003; TEO: P = 0.004; TE: P = 0.030;LIP: P = 0.028; FEF: P = 0.019; VLPFC: P = 0.006; OFC: P =0.008; amygdala: P < 0.001; but not V3: P = 0.898; V4: P = 0.596;DLPFC: P = 0.128).After OT administration, reduced responses to aggressive

faces relative to neutral faces were found in half of the face-responsive ROIs (V4: P = 0.017; TEO: P = 0.032; LIP: P = 0.014;FEF: P = 0.051; DLPFC: P = 0.032; VLPFC: P = 0.006). Sig-nificant or nearly significant interactions between treatment andvalence [(aggressive vs. neutral in the placebo condition) vs.(aggressive vs. neutral in the OT condition)] were found in manybut not all of the face-responsive ROIs (V1: P = 0.005; V2: P =0.119; V3: P = 0.848; V4: P = 0.042; TEO: P = 0.063; TE: P =0.156; LIP: P = 0.043; FEF: P = 0.120; DLPFC: P = 0.021;VLPFC: P = 0.069; OFC: P = 0.645; amygdala: P = 0.062). Theseinteractions indicate that OT reduced the response evoked byaggressive relative to neutral faces in a large proportion of theface-responsive ROIs. Similar to fearful faces, the effect of OTon the valence effect for aggressive faces was mainly driven byreduced responses evoked by aggressive, rather than neutral,faces (aggressive faces in the placebo condition vs. in the OT

condition: V1: P < 0.001; V2: P = 0.037; TEO: P = 0.022; LIP:P = 0.013; FEF: P = 0.051; amygdala: P = 0.017; but not V3: P =0.549; V4: P = 0.797; TE: P = 0.347; DLPFC: P = 0.116; VLPFC:P = 0.095; OFC: P = 0.427).As found in the placebo condition, enhanced responses to

appeasing faces relative to neutral faces were still present in thesame set of face-responsive ROIs (V4: P = 0.039; TEO: P =0.013; TE: P < 0.001; LIP: P < 0.001; FEF: P < 0.001; DLPFC: P <0.001; VLPFC: P < 0.001; amygdala: P = 0.011), except V1 (P =1.000), after OT administration. Similar to neutral faces, OTadministration did not significantly alter the response to ap-peasing faces. No significant interactions between treatment andvalence [(appeasing vs. neutral in the placebo condition) vs.(appeasing vs. neutral in the OT condition)] were found in any ofthe defined ROIs, indicating that OT did not modulate the va-lence effect for appeasing faces.

Functional Connectivity. An ANOVA with repeated measuresfound a significant main effect for ROI pair [F (65, 11,310) =555.782; P < 0.001], but not expression [F (3, 522) = 0.268; P =0.849] or treatment [F (1, 174) = 1.117; P = 0.292]. There weresignificant interactions between ROI pair and expression [F (195,33,930) = 1.186; P = 0.039] and between ROI pair and treatment[F (65, 11,310) = 4.377; P < 0.001], but not between expressionand treatment [F (3, 522) = 1.603; P = 0.188]. Moreover, the

Fig. 2. Averaged fMRI responses across all three subjects to various facialexpressions within areas in the occipital and inferior temporal cortex in theplacebo and OT conditions. Asterisks on histograms indicate a significantdifference between emotional and neutral faces within treatment (*P <0.05, **P < 0.01, ***P < 0.001) or a significant interaction between treat-ment and valence (★P < 0.05, ★★P < 0.01). FG, fear grin (fearful); N, neutral;LS, lip smack (appeasing); T, threat (aggressive).

Fig. 3. Averaged fMRI responses across all three subjects to various facialexpressions within subregions of the PFC (FEF, DLPFC, VLPFC and OFC), LIP,and the amygdala in the placebo and OT conditions. Asterisks on histogramsindicate a significant difference between emotional and neutral faces withintreatment (*P < 0.05, **P < 0.01, ***P < 0.001) or a significant interactionbetween treatment and valence (★P < 0.05, ★★P < 0.01).

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interaction among ROI pair, expression, and treatment wassignificant [F (195, 33,930) = 1.181; P = 0.043], indicating thatthe effect of OT on functional coupling evoked by facial ex-pressions differed across ROI pairs.

Placebo Condition. For each facial expression, significant pairwisecorrelations were found for each ROI pair (Fig. 4). However,there were no significant differences among the functional con-nectivity maps (FCMs) for different facial expressions.

OT Condition. Because the three-way interaction (treatment ×expression × ROI pair) was significant, analyses were conductedfor each ROI pair with one within-subject factor (expression),one between-subject factor (treatment), and one nuisance factor(monkey). Only results that showed a significant interactionbetween expression and treatment for a given ROI pair arepresented here.Significant interactions between treatment and fearful versus

neutral faces [(fearful vs. neutral in the placebo condition) vs.(fearful vs. neutral in the OT condition)] were found between theamygdala and areas in the occipital and inferior temporal cortex(amygdala–V1: P = 0.042; amygdala–V3: P = 0.022; amygdala–V4: P = 0.026; amygdala–TEO: P = 0.035; amygdala–TE: P =0.022), with the exception of amygdala–V2 (P = 0.128). Asshown in Fig. 5B, significant interactions were mainly induced bythe reduced functional coupling (coded by warm colors) amongthese areas in the signal evoked by fearful faces after OT ad-ministration (fearful faces in the placebo condition vs. in the OTcondition: amygdala–V1: P = 0.001; amygdala–V3: P = 0.009).Significant interactions between treatment and aggressive

versus neutral faces [(aggressive vs. neutral in the placebo con-dition) vs. (aggressive vs. neutral in the OT condition)] were alsofound between the amygdala and areas in the occipital and in-ferior temporal cortex (amygdala–V2: P = 0.026; amygdala–V3:P = 0.040; amygdala–V4: P = 0.005; amygdala–TEO: P = 0.009;amygdala–TE: P = 0.017) with the exception of amygdala–V1(P = 0.251). We similarly found significant interactions betweensubregions of the PFC (particularly VLPFC) and areas in theoccipital and inferior temporal cortex (VLPFC–V3: P = 0.014;VLPFC–V4: P = 0.020; VLPFC–TEO: P = 0.020; OFC–V3: P =0.028), as well as between the occipital and inferior temporalcortex (V2–TEO: P = 0.027; V2–TE: P = 0.032; V3–TEO: P =0.030; V3–TE: P = 0.008; V4–TEO: P = 0.017). As observed forfearful faces, these interactions were mainly induced by the re-duced functional coupling (coded by warm colors in Fig. 5C)among these areas in the signal evoked by aggressive faces afterOT administration (aggressive faces in the placebo condition vs.in the OT condition: amygdala–V2: P = 0.026; amygdala–V3: P =0.012; amygdala–V4: P = 0.048; amygdala–TE: P = 0.038; V2–TEO: P = 0.001; V2–TE: P = 0.002; V3–TEO: P = 0.001; V3–TE:P = 0.003; V4–TEO: P = 0.031).The interactions between treatment and appeasing versus

neutral faces [(appeasing vs. neutral in the placebo condition) vs.(appeasing vs. neutral in the OT condition)] were only presentfor one ROI pair (TEO–DLPFC: P = 0.040), which was caused bythe reduced functional coupling in the signal evoked by appeasingfaces after OT administration (P = 0.003); no systemic pattern forappeasing faces was found similar to the ones observed for fearfuland aggressive faces (Fig. 5D). This result indicates that OT ad-ministration had less of an effect on functional coupling betweenface-responsive ROIs in the signal evoked by appeasing facescompared with those evoked by fearful and aggressive faces.

DiscussionIn the present study, we investigated the effects of OT on fMRIresponses evoked by different facial expressions (neutral, fearful,aggressive, and appeasing) in face-responsive ROIs within theoccipital cortex, inferior temporal cortex, area LIP of parietal

cortex, PFC, and amygdala of monkeys. We found that OT didnot alter the fMRI response to neutral faces; OT differentiallyaltered responses to emotional faces, significantly reducing re-sponses to both fearful and aggressive faces while not changingthe response to appeasing faces; and OT reduced functionalcoupling between the amygdala and areas in the occipital andinferior temporal cortex selectively in response to fearful andaggressive faces, but not neutral or appeasing faces. Here, wediscuss the significance of these findings to understand the un-derlying neural mechanisms of the effects of OT.

OT Modulates Brain Activity in Multiple Neural Systems. In the pre-sent study, in addition to the amygdala, we selected ROIs in theventral visual pathway (from V1 to area TE), which respondselectively to visual features relevant for object identification,such as color, shape, and texture (27). We also investigated theeffects of OT on brain activity in FEF and LIP, which are heavilyinterconnected and important for visual attention (28). Finally,we examined OT’s modulatory effects on three subregions of thePFC: DLPFC, VLPFC, and OFC (29), which are areas knownto play important roles in emotion-related cognitive behavior.For example, OFC damage is associated with deficits in facialexpression recognition (30); VLPFC is sensitive to changes in fa-cial features, expressions, and gaze direction (31); and DLPFC isthought to play a central regulatory role in emotion processing(32). Similar modulatory effects of OT were found in all theseROIs: OT differentially altered responses to emotional faces.Thus, we provide the first evidence in monkeys, to our knowl-edge, that intranasally administered OT modulates brain activityin multiple neural systems, including those mediating visualperception (the occipital and inferior temporal cortex), emotion(the amygdala), attention (LIP and FEF), and higher cognitivefunction (PFC). Our findings are consistent with human neuro-imaging results. Kirsch et al. found that OT reduces responses to

Fig. 4. FCMs averaged across all three subjects in the placebo condition. TheFCMs evoked by neutral, fearful, aggressive, and appeasing faces are shownin A, B, C, and D, respectively. The color code indicates the level of partialpairwise correlation coefficients among the ROIs. IT, inferior temporal cor-tex; OC, occipital cortex.

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fear-inducing stimuli in the amygdala (15). Domes et al. alsofound that OT attenuates activation in the amygdala, as well asin frontal and temporal areas, for angry and fearful faces (16).Furthermore, our findings may help explain OT’s modulatoryeffects on social behavior and cognition in monkeys. For exam-ple, a recent study in monkeys found that intranasal OT miti-gates the influence of emotional faces on attention (23), whichmay reflect the effect of OT on brain structures involved inemotion (the amygdala) and attention (FEF and LIP) found inthe present study. Our findings demonstrate homologies betweenmonkeys and humans in the neural circuits mediating the effectsof OT and provide key information for understanding the be-havioral consequences of OT administration in monkeys.

OT Reduces Brain Activity Evoked by Negative Emotional Stimuli.Here, we demonstrate that intranasally administered OT inmonkeys reduces activity in face-responsive ROIs to fearful andaggressive faces, but not to neutral or appeasing faces. Consis-tent with our findings, a previous study found that intranasallyadministered OT reduced monkeys’ attention to negative facialexpressions (fear grin and open-mouth threat), but not to neutralor nonsocial stimuli (24).Although our results showed robust effects of OT on fearful

and aggressive faces, they also showed that OT did not signifi-cantly alter the valence effect elicited by appeasing faces in theplacebo condition. The appeasing faces we used, those express-ing lip smack, could be considered an affiliative expression (33).It is possible that the lack of effect seen for appeasing faces inour study relates to the inconsistent results reported in humansduring the viewing of happy faces (another affiliative expression)

after OT administration. For instance, Domes et al. reportedthat OT reduces the amygdala fMRI activation evoked by happyfaces (16), whereas another study found enhanced responses tohappy faces after OT administration (3). The discrepancy be-tween our results and human findings may also be rooted indifferences between the meaning of lip smack in macaques and ahappy expression in humans.In the placebo condition, unlike fearful faces, aggressive faces

did not evoke stronger fMRI responses relative to neutral faces.As suggested previously (34), the individual variability in theneural response to an aggressive facial expression (open-mouththreat) may depend on the animal’s rank in the social hierarchy.However, regardless of the difference between fearful and ag-gressive faces in the placebo condition, OT had a similar mod-ulatory effect on responses to fearful and aggressive faces, whichwere both reduced. These distinct findings suggest that theneural mechanisms underlying the effect of OT on responses tofearful and aggressive faces may differ. In support of this idea,although we found that the effect of OT on responses to fearfuland aggressive faces was similar across the different ROIs, OTadministration differentially altered FCMs evoked by fearful andaggressive faces: reducing functional coupling between the PFC(especially VLPFC) and areas in the occipital and inferiortemporal cortex in the signal evoked by aggressive faces, but notfearful faces.In summary, our findings support the idea that OT does not

affect the perception of all social stimuli in the same way but,instead, selectively affects the perception of negative (fearful andaggressive) emotional stimuli. Importantly, our results demon-strate homologies between monkeys and humans not only in theneural circuits mediating the effects of OT but also in the un-derlying neural mechanisms of OT’s effects on social behaviorand cognition. The results thus support the idea that the monkeyis an ideal animal model to further explore the development ofOT-based pharmacological strategies to treat patients with dys-functional social behavior.

How Does OT Modulate the Face-Responsive Network? In the pre-sent study, we found that the amygdala and many face-responsivecortical areas, including the occipital cortex, inferior temporalcortex, parietal cortex, and PFC, exhibited similar patterns ofmodulation after OT administration. These modulatory effectscould be driven by two, not mutually exclusive mechanisms.First, previous studies have demonstrated that intranasally

administered OT could enter the central nervous system (20, 35,36) and then bind to OT receptors there. In the rhesus monkeybrain, OT receptors are most robustly expressed in the nucleusbasalis of Meynert, the superficial gray layer of the superiorcolliculus, the pedunculopontine tegmental nucleus, the trape-zoid body, and the ventromedial hypothalamus (37). Although (ahigh density of) OT receptors have not been found in the se-lected ROIs of the present study, including the amygdala, activityin these ROIs may be modulated by projections from areas thatcontain OT receptors, and thereby influence social behavior andcognition. For example, the nucleus basalis of Meynert providesheavy cholinergic inputs to both the basolateral amygdala andcerebral cortex (38, 39).On the other hand, OT is closely related to vasopressin (AVP)

and can bind to AVP receptors (vasopressin 1a, AVP1A) (40).Studies in rodents have found that the effects of OT on somesocial behavior and cognition (e.g., social communication) aremediated by AVP1A receptors (41, 42). AVP1A receptors aredensely located in the prefrontal, cingulate, pyriform, andentorhinal cortex, as well as the presubiculum and mammillarybodies, but are also found in the amygdala, bed nucleus of thestria terminalis, lateral septum, hypothalamus, and brainstem(43). Two of these areas were selected as ROIs in the presentstudy, the amygdala and PFC, which exhibited a pattern of

Fig. 5. Alteration in FCMs caused by OT administration. Differences be-tween the placebo and OT conditions (FCMs in the placebo condition − FCMsin the OT condition) in partial pairwise correlation coefficients evoked byneutral, fearful, aggressive, and appeasing faces are shown in A, B, C, and D,respectively. The color code indicates the level of these differences. Thedashed lines highlight functional coupling between the amygdala and areasin the occipital and inferior temporal cortex. Asterisks on matrices indicate asignificant interaction between treatment and emotional faces versus neu-tral faces [(emotional vs. neutral in the placebo condition) vs. (emotional vs.neutral in the OT condition); *P < 0.05, **P < 0.01].

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modulation after OT administration similar to the other selectedROIs. Neuroanatomical studies in monkeys have revealed thatthe amygdala and PFC are interconnected with the other se-lected ROIs (44–48). For example, feedback projections fromthe amygdala terminate in virtually all areas in the inferiortemporal and occipital cortex, including V1 (44). Therefore, theobserved effects of OT in the present study could be mediated byAVP1A receptors in the amygdala and/or PFC. This is consistentwith our findings that the other selected ROIs showed similarvalence effects to those observed in the amygdala and PFC in theplacebo condition (e.g., fear grin consistently elicited the greatestresponse), that the reductions of activity by OT were similar inall of the selected ROIs [e.g., OT selectively affected the pro-cessing of negative (fearful and aggressive) emotional stimuli],and that the functional coupling between the amygdala/PFC andinferior temporal/occipital cortex evoked by negative (fearfuland aggressive) emotional faces was reduced after OT adminis-tration. Thus, the observed alterations in brain activity during theperception of facial expressions after OT administration may bemediated by AVP receptors, and thus may not be specific to OT;that is, AVP may cause similar effects.It should be noted that OT might also modulate social be-

havior and cognition through interactions with both OT and AVPreceptors. Future studies (e.g., local injection of OT into specificareas that contain OT receptors) are needed to further explore theneural mechanisms underlying OT’s modulatory effects.Taken together, our results suggest that activity evoked by

emotional stimuli, especially negative stimuli, is systematicallyaltered after OT administration within multiple neural systems,including those mediating visual perception (the occipital andinferior temporal cortex), emotion (the amygdala), attention(LIP and FEF), and higher cognitive function (PFC). In addi-tion, our results demonstrate homologies between human andmonkey in the neural circuits mediating the effects of OT,thereby supporting the use of the nonhuman primate as an an-imal model for further studies of OT’s effects. We anticipate thatthe results of such studies will deepen our understanding of howthis neuropeptide affects the brain and, eventually, facilitatehuman clinical applications.

Experimental ProceduresSubjects and General Procedures. Three male macaque monkeys (monkeys I,D, and P;Macaca mulatta; 9 y old; 6.5–7.5 kg) were used. They were acquiredfrom the same primate breeding facility in the United States, where theyhad social group histories as well as group-housing experience until theirtransfer to the National Institute of Mental Health (NIMH) for quarantine atthe age of approximately 4 y. After that, they were individually caged withauditory and visual contact with other conspecifics in the same colony room,which accommodates about 20 rhesus monkeys. All three animals used inthis study had been housed at NIMH for 4–5 y before this experiment. Thus,all three subjects have had extensive social experience, which made themfamiliar with perception and interpretation of facial cues in conspecifics.

All procedures followed the Institute of Laboratory Animal Research (part ofthe National Research Council of the National Academy of Sciences) guidelinesand were approved by the NIMH Animal Care and Use Committee. Eachmonkey was surgically implanted with a magnetic resonance (MR)-compatiblehead post under sterile conditions, using isoflurane anesthesia. After recovery,subjects were trained to sit in a plastic restraint chair and fixate a central targetfor long durations with their heads fixed, facing a screen on which visualstimuli were presented (49, 50).

Brain Activity Measurements. Functional and anatomical MRI scanning wascarried out in the Neurophysiology Imaging Facility Core [NIMH, NationalInstitute of Neurological Disorders and Stroke (NINDS), National Eye Institute(NEI)]. Before each scanning session, an exogenous contrast agent [mono-crystalline iron oxide nanocolloid (MION)] was injected into the femoral orexternal saphenous vein (12–15 mg/kg) to increase the contrast/noise ratioand to optimize the localization of fMRI signals (51, 52). Imaging data werecollected in a 4.7 T Bruker scanner with a surface coil array (eight elements).Twenty-eight 1.5-mm coronal slices (no gap) were acquired using single-shot

interleaved gradient-recalled echo planar imaging. Imaging parameterswere as follows: voxel size: 1.5 mm isotropic, field of view: 96 × 54 mm;matrix size: 64 × 36; echo time (TE): 13.8 ms; repetition time (TR): 2 s; flipangle: 90°. A low-resolution anatomical scan was also acquired in the samesession to serve as an anatomical reference (modified driven equilibriumFourier transform sequence, voxel size: 1.5 × 0. 5 × 0.5 mm; field of view: 96 ×96 mm; matrix size: 128 × 128; TE: 2.932 ms; TR: 6.24 ms; flip angle: 12°). Tofacilitate cortical surface alignment, we acquired high-resolution T1-weightedwhole-brain anatomical scans in separate sessions, using the modified drivenequilibrium Fourier transform sequence. Imaging parameters were as follows:voxel size: 0.5 mm isotropic; TE: 4.9 ms; TR: 13.8 ms; flip angle: 14°.

Experimental Design and Task. All stimuli used in this experiment wereidentical to the ones used in Hadj-Bouziane et al. (34, 49). Briefly, the stimuliwere color images of facial expressions displayed by eight unfamiliar ma-caque monkeys (frontal view): neutral, fearful (fear grin), aggressive (open-mouth threat), and appeasing (lip smack). We presented these four differentfacial expressions to each monkey in a block design using Presentationsoftware (version 12.2, www.neurobs.com). Stimuli spanned a visual angle of11° (maximal horizontal and/or vertical extent) and were presented foveallyfor 700 ms on a uniform gray background, with a fixation square (0.2° in red)superimposed on each image, followed by a 300-ms blank period. Stimulifrom each facial expression were presented in blocks of 32 s each, in-terleaved with 20-s fixation blocks (neutral gray background). Individualruns began and ended with a fixation block. Each categorical block waspresented twice in each run in a pseudorandom order. Different pseudo-random sequences were used in each run.

To locate the ROIs, we also performed an independent functional localizerexperiment in all three animals, using the same fMRI parameters as in themain experiment, but with a different set of stimuli. In the localizer exper-iment, the stimuli were presented in a block design. Grayscale photos ofneutral monkey faces, Fourier-phase scrambled faces, familiar places, andfamiliar objects were presented in categorical blocks. Each block lasted 40 s,during which each of 20 images was presented for 2 s, alternating with 20-sfixation blocks (neutral gray background). Individual runs began and endedwith a fixation block. Each categorical block was presented twice in each run.Each of the three monkeys was scanned in one localizer session, resulting in atotal of 15–16 runs per monkey.

In both the facial expression and localizer experiments, the monkeys wererequired to maintain fixation on a square superimposed on the stimuli toreceive a liquid reward. In the reward schedule, the frequency of rewardincreased as the duration of fixation increased (49, 50). Eye position wasmonitored with an infrared pupil tracking system (iView, Inc).

Intranasal OT Administration. Intranasal administration has been widely usedto examine the effects of OT on social behavior and cognition. Severalprevious studies have studied which OT delivery method elevates cerebro-spinal fluid OT concentrations in rhesus macaques (20, 35, 36). The methodused in the present study has been demonstrated to be effective in causingelevations of OT concentration in cerebrospinal fluid (35). Before beginningthe experiment, the monkeys were habituated to receiving saline intranasalspray using atomizers (Intranasal Mucosal Atomization Device; Wolfe ToryMedical) attached to syringes (35). The animals’ heads were fixed during thisprocedure to minimize movement and enhance the reliability of dosing. Thishabituation procedure was repeated until the monkeys were completelyrelaxed during the nasal spray administration.

On the day of the experiment, monkeys were transported in a primatechair from the colony room to the scanning room. After fixing their heads,subjects were administered intranasally either OT (Sigma, 24 IU) or placebo(sterile saline) in a 0.4-mL volume. This is similar to the dose previously foundto affect socially relevant behavior in monkeys (e.g., refs. 20–22) and humans(e.g., refs. 8, 15, 16). Scanning began about 40 min after each treatment. A40-min delay between drug administration and the start of scanning issimilar to the timing used in previous monkey and human studies (20–22,53), as elevations of OT concentration in cerebrospinal fluid have beenfound ∼40 min after OT administration in monkeys (20, 35, 36) and humans[AVP, similar to OT with only a two-amino acid difference (54)]. The threemonkeys were scanned in two separate sessions per treatment (placebo orOT), resulting in a total of 30 runs (60 condition repetitions) per monkey pertreatment. The order of treatments was randomized for each monkey, withat least 5 d intervening.

Data Analysis.fMRI data preprocessing. Functional data were preprocessed using Analysis ofFunctional NeuroImages software (AFNI) (55). All runs were concatenated

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across all sessions. Images were realigned to the mean volume of thelocalizer session. The data were smoothed with a 2-mm full-width half-maximum Gaussian kernel. Signal intensity was normalized to the meansignal value within each run. For each voxel in both experiments, we per-formed a single univariate linear model fit to estimate the response ampli-tude for each condition. The model included a hemodynamic responsepredictor for each condition and regressors of no interest [baseline, move-ment parameters from realignment corrections, and signal drifts (linear aswell as quadratic)]. A general linear model and a MION kernel were used tomodel the hemodynamic response function (56).Definition of face-responsive ROIs. All ROIs were defined based on the localizerexperiment. For each monkey, face-responsive voxels corresponded to voxelssignificantly more active for neutral faces compared with a blank screen (P <10−4 uncorrected). Therein, we identified the following anatomically de-fined areas as described in the Saleem and Logothetis stereotaxic atlas (57):the occipital cortex: V1, V2, V3, and V4; the inferior temporal cortex: theposterior portion (area TEO) and the anterior portion (area TE); the amyg-dala; the LIP; and the PFC: FEF, DLPFC, VLPFC, and OFC.

V1 included the posterior half of operculum and the ascending anddescending limbs of the calcarine sulcus, as well themost posterior part of thestem of this sulcus. We defined V2 on the ventral surface in the lip of thelower bank of the calcarine sulcus, including a small portion of the medialsurface near the upper bank of this sulcus, and in islands of graymatter withinthe posterior portions of the lunate and parieto-occipital sulci. V3 was de-fined as a narrow strip of cortex, located immediately anterior to V2 andposterior to V4 both dorsally and ventrally in the hemisphere. V4 extendedbetween the lunate sulcus and the sts on the prelunate gyrus and extendingventrally through the anterior bank of the inferior occipital sulcus, as pre-viously described (58). We defined TEO as extending from just anterior to theinferior occipital sulcus rostrally for 1 cm, dorsally to include the fundus andventral bank of the sts, and ventromedially to include the lateral bank of theoccipitotemporal sulcus TE was defined as adjacent and rostral to TEO,extending to the tip of the temporal pole, dorsally to include the fundus andventral bank of the sts, and ventromedially to include the lateral bank of theoccipitotemporal sulcus. LIP included both the dorsal and ventral subregionswithin the lateral bank of the inferior parietal sulcus (59). PFC extendedrostrally from the fundus of the arcuate sulcus to include the lateral, medial,and orbital cortex. Within the PFC, we delineated DLPFC as extending fromthe fundus of the principal sulcus medially to the dorsal lip of the cingulatesulcus, VLPFC as extending from the fundus of the principal sulcus ventrallyto the lateral edge of the ventral surface, and OFC as adjacent to VLPFC,extending medially to the medial edge of the ventral surface. FEF was de-fined within the rostral bank of the genu of the arcuate sulcus, extendinganteriorly over the sulcal lip. The extent of the amygdala was determinedfrom high-resolution structural MRI scans.

The other localizer comparisons (e.g., faces vs. objects) resulted in morecircumscribed regions (e.g., face-selective regions) locatedwithin the selected

face-responsive ROIs. These regions did not show significant differences inresponse properties in both placebo and OT condition, relative to ROIs thatincluded them.Responses to neutral and emotional faces. The signal in the facial expressionexperiment was extracted from face-responsive ROIs. We then calculated theresponse to each facial expression within each run (averaged across tworepetitions) and performed ANOVAs with repeated measures for two within-subject factors (ROI and expression) and one between-subject factor (treat-ment), followed by post hoc analyses and tests for interactions. P values wereBonferroni corrected for the number of comparisons. For all of the ANOVAs,we included the monkey as the nuisance between-subject factor. This pro-cedure allowed us to test for our factors of interest (described earlier) whilealso statistically controlling for nuisance variability among monkeys.Functional connectivity. To investigate the context-specific changes in func-tional connectivity, the median activity within each face-responsive ROI wasextracted for each condition. The points selected for each condition werebased on the MION response model (i.e., convolving the MION kernel with asquare wave of 32 s, then scaled to have a magnitude of 1). To reduce thenoise level of these time series, only points in which the amplitude of theresponse model was greater than 0.1 were included for each condition (i.e.,30 of 32 points were selected). Then a pairwise correlation was calculatedbetween each pair of ROIs. To rule out correlations caused by the shared taskinput and motion, the response model and movement parameters werecontrolled (i.e., via partial correlations). The partial correlation coefficientswere calculated for each facial expression within each run (concatenating thetwo repetitions). We then applied Fisher’s z transformation to these corre-lation coefficients to convert them into normally distributed variables suit-able for parametric statistical testing. Similar ANOVAs to those for responsesto neutral and emotional faces were conducted: two within-subject factors(ROI pair and expression) and one between-subject factor (treatment), fol-lowed by multiple post hoc analyses and tests for interactions. P values wereBonferroni corrected for the number of comparisons. We also included themonkey as a nuisance between-subject factor. Note that the values shown inFigs. 4 and 5 depict the raw, untransformed correlation values, which werenot included in any statistical analysis.

As previously reported, there were no systematic significant differencesbetween hemispheres in the response profile of any of the three monkeys(34). Thus, the analyses shown in the Results were based on pooled datafrom both hemispheres. Moreover, results from the individual monkeyswere similar. Thus, we present results averaged across all three monkeys.

ACKNOWLEDGMENTS. This work was supported by the National Institute ofMental Health Intramural Research Program (N.L., F.H-B., K.B.J., J.N.T., B.B.A.,and L.G.U.). We thank Frank Q. Ye, Charles C. Zhu, and David C. Ide fortechnical assistance; Katalin M. Gothard for providing the original monkeyfacial expression stimuli; and Biying Xu for providing the exogenouscontrast agent.

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