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

Section on Neurocircuitry, Laboratory of Brain and Cognition, NIMH, NIH, Bethesda, MD 20892; bSection on Cognitive Neuroscience, Laboratory of Neuropsychology, 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)

| neuroimaging | nonhuman primate | social stimuli

Significance

I

n the last decade, oxytocin (OT), a mammalian hormone, has become one of the most studied peptides of the neuroendocrine system. In humans, accumulating evidence has demonstrated that OT affects a wide range of social behavior and cognition, including perception, recognition and memory of social stimuli (1–5), socially reinforced learning (6), and more complex 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 for various disorders with dysfunctional social behavior, such as autism spectrum disorders, antisocial personality disorder, and schizophrenia (for review, see ref. 12). A recent study found that OT enhances brain activity for socially meaningful stimuli but attenuates activity for nonsocially meaningful stimuli in children with autism spectrum disorders (13). Although these studies suggest very promising prospects of OT for clinical use, the neural mechanisms underlying OT’s modulatory effects remain elusive. To understand these mechanisms, it is important to investigate the effect of OT on brain activity, especially in regions involved in social behavior and cognition. Functional magnetic resonance imaging (fMRI) has been the major approach to investigating altered brain activation patterns in response to OT in humans. OT may affect the perception of social stimuli, and thus mediate subsequent social information processing (e.g., learning and memory, etc.) (14). Many fMRI studies have examined the effects of OT on brain activity during www.pnas.org/cgi/doi/10.1073/pnas.1508097112

Oxytocin (OT), a mammalian hormone, may serve as a treatment for psychiatric disorders because of its beneficial effect on social behavior. Here, we found that in monkeys, OT selectively altered brain activity within multiple neural systems (visual perception, emotion, attention, and higher cognition function) and functional coupling between the amygdala and areas in the ventral visual pathway evoked by negative emotional expressions. Our findings provide key information for understanding the behavioral consequences of OT administration and indicate homologies between monkeys and humans in the neural circuits mediating the effects of OT. Thus, the monkey may be an ideal animal model to explore the development of OT-based pharmacologic strategies for treating patients 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., and J.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. 1

To whom correspondence may be addressed. Email: [email protected] or ungerlel@ mail.nih.gov.

2

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

3

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

PNAS | Published online May 26, 2015 | E3123–E3130

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the perception of social stimuli to probe the brain regions that underlie OT’s modulatory effects. Emotional stimuli, which are crucial for social communication and interaction, have been mainly used. For example, Kirsch et al. showed that OT reduces activation in response to fear-inducing stimuli in the amygdala, a key brain region involved in emotional regulation (15). Subsequently, a series of studies examined the effects of OT on responses to facial expressions (3, 16), to conditioned facial expressions (17), and to threatening scenes (18). These studies showed that activity evoked by emotional stimuli, especially negative stimuli (e.g., fearful faces, but see refs. 3 and 16 for happy faces), is systematically altered within an interconnected network of brain regions after OT administration. Because of the limitation of experimental approaches with human subjects, animal models are essential not only for investigating the neural mechanisms underlying the effects of OT but also for exploring OT-based therapeutic strategies for individuals with dysfunctional social behavior. Given the similarities between monkeys and humans in the neural circuitry underlying social cognition (19), the rhesus macaque could be an ideal animal model to examine the effects of OT. To date, only a few studies have investigated the behavioral consequences of OT administration in monkeys (20–25). Consistent with the human literature, these studies have found that intranasal administration of OT affects social behavior and cognition in monkeys,

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Increasing evidence has shown that oxytocin (OT), a mammalian hormone, modifies the way social stimuli are perceived and the way they affect behavior. Thus, OT may serve as a treatment for psychiatric disorders, many of which are characterized by dysfunctional social behavior. To explore the neural mechanisms mediating the effects of OT in macaque monkeys, we investigated whether OT would modulate functional magnetic resonance imaging (fMRI) responses in face-responsive regions (faces vs. blank screen) evoked by the perception of various facial expressions (neutral, fearful, aggressive, and appeasing). In the placebo condition, we found significantly increased activation for emotional (mainly fearful and appeasing) faces compared with neutral faces across the faceresponsive regions. OT selectively, and differentially, altered fMRI responses to emotional expressions, significantly reducing responses to both fearful and aggressive faces in face-responsive regions while leaving responses to appeasing as well as neutral faces unchanged. We also found that OT administration selectively reduced functional coupling between the amygdala and areas in the occipital and inferior temporal cortex during the viewing of fearful and aggressive faces, but not during the viewing of neutral or appeasing faces. Taken together, our results indicate homologies between monkeys and humans in the neural circuits mediating the effects of OT. Thus, the monkey may be an ideal animal model to explore the development of OT-based pharmacological strategies for treating patients with dysfunctional social behavior.

Fig. 1. fMRI response to neutral faces compared with a blank screen in the localizer experiment and responses to emotional faces compared with neutral faces 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 of the right hemisphere of the inflated cortex (Top) and on coronal slices through the amygdala (Bottom) of monkey P. The locations of the coronal slices are marked by the black line on the lateral views. The color bars show the statistical values of the contrast between either neutral faces and a blank screen or between emotional and neutral faces. as, arcuate sulcus; ios, inferior occipital sulcus; ls, lateral sulcus; lus, lunate sulcus; pmts, posterior middle temporal sulcus; sts, superior temporal sulcus.

including vicarious as well as self-reinforcement (20), social vigilance (22), socially reinforced learning (26), and attention to facial features and expressions (21, 24). However, how OT exerts its effects on brain activity in monkeys remains unclear. To explore the neural mechanisms mediating the effects of OT in macaque monkeys, in the present study, we investigated whether OT would modulate fMRI responses evoked by the perception of 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 different conditions: placebo control (saline) and intranasal OT. We predicted that in the placebo condition, emotional faces (especially fearful) would evoke enhanced activation compared with neutral faces, that is, they would show a valence effect; that, as in humans, OT administration would reduce this valence effect in monkeys; and that OT administration would alter functional coupling among those brain regions showing a valence effect. Results Responses to Neutral and Emotional Faces. Using the contrast of neutral faces versus a blank screen in the localizer experiment, we found that face-responsive voxels were widely distributed bilaterally within the occipital cortex (V1, V2, V3, V4), within the lateral intraparietal sulcus (LIP), across the anterior portion (area TE) and the posterior portion (area TEO) of the inferior temporal cortex, within and along the superior temporal sulcus (sts), within the dorsal portion of the lateral and basal nuclei of the amygdala, and within the prefrontal cortex (PFC), including the frontal eye field (FEF), dorsolateral prefrontal cortex (DLPFC), ventrolateral prefrontal cortex (VLPFC), and orbitofrontal cortex (OFC) in all three subjects (Fig. 1A). Using the contrast of emotional faces versus neutral faces, we found a similar, although spatially more limited, activation map (Fig. 1B). We performed an ANOVA with two within-subject factors [region of interest (ROI) and expression] and one betweensubject factor (treatment: placebo vs. OT). We found significant main effects for ROI [F (11, 1,914) = 2,580.668; P < 0.001] and expression [F (3, 522) = 44.010; P < 0.001], but not for treatment [F (1, 174) = 1.867; P = 0.174]. There were significant interactions 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 E3124 | www.pnas.org/cgi/doi/10.1073/pnas.1508097112

significant [F (33, 5,742) = 0.997; P = 0.473]. These findings indicate that OT differentially altered fMRI responses to facial expressions, and the effect was similar across face-responsive ROIs. To provide a complete picture of the effects of OT on responses to neutral and emotional faces, we present the results from each ROI in detail by conducting post hoc analyses and tests for interactions, aware that the three-way interaction among ROI, expression, and treatment was not significant. Placebo Condition. We evaluated responses to various facial expressions by contrasting each category of emotional faces (fearful, aggressive, and appeasing) with neutral faces. This analysis showed, relative to neutral faces, in all face-responsive ROIs, enhanced responses to fearful faces (P < 0.001) and appeasing faces (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 aggressive faces did not significantly differ from those to neutral faces in the defined ROIs. The group-averaged response profiles for each ROI in the placebo condition are illustrated in Figs. 2 and 3. OT Condition. OT does not alter the responses to neutral faces. We first investigated

the effect of OT on the fMRI signal evoked by neutral faces. We found no difference in the response to neutral faces between the OT and placebo conditions in any of the face-responsive ROIs, indicating that OT administration did not affect neutral face processing (Figs. 2 and 3). OT modulates the valence effect. After OT administration, enhanced responses to fearful faces relative to neutral faces observed in the placebo condition were no longer present in V1 (P = 0.828), the amygdala (P = 1.000), or OFC (P = 0.275). Although enhanced responses to fearful faces were still present in the other faceresponsive ROIs after OT administration, significant or nearly significant interactions between treatment and valence [(fearful vs. neutral in the placebo condition) vs. (fearful vs. neutral in the OT 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 the enhanced response to fearful relative to neutral faces (i.e., reduced the valence effect for fearful faces). Because OT did not alter the response to neutral faces, OT administration mainly Liu et al.

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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 the same 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, OT administration did not significantly alter the response to appeasing faces. No significant interactions between treatment and valence [(appeasing vs. neutral in the placebo condition) vs. (appeasing vs. neutral in the OT condition)] were found in any of the defined ROIs, indicating that OT did not modulate the valence effect for appeasing faces. Functional Connectivity. An ANOVA with repeated measures found 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 were significant 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 expression and treatment [F (3, 522) = 1.603; P = 0.188]. Moreover, the

Liu et al.

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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 faceresponsive 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). Significant or nearly significant interactions between treatment and valence [(aggressive vs. neutral in the placebo condition) vs. (aggressive vs. neutral in the OT condition)] were found in many but 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). These interactions indicate that OT reduced the response evoked by aggressive relative to neutral faces in a large proportion of the face-responsive ROIs. Similar to fearful faces, the effect of OT on the valence effect for aggressive faces was mainly driven by reduced responses evoked by aggressive, rather than neutral, faces (aggressive faces in the placebo condition vs. in the OT

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Fig. 2. Averaged fMRI responses across all three subjects to various facial expressions within areas in the occipital and inferior temporal cortex in the placebo and OT conditions. Asterisks on histograms indicate a significant difference between emotional and neutral faces within treatment (*P < 0.05, **P < 0.01, ***P < 0.001) or a significant interaction between treatment 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 facial expressions within subregions of the PFC (FEF, DLPFC, VLPFC and OFC), LIP, and the amygdala in the placebo and OT conditions. Asterisks on histograms indicate a significant difference between emotional and neutral faces within treatment (*P < 0.05, **P < 0.01, ***P < 0.001) or a significant interaction between treatment and valence (★P < 0.05, ★★P < 0.01).

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interaction among ROI pair, expression, and treatment was significant [F (195, 33,930) = 1.181; P = 0.043], indicating that the effect of OT on functional coupling evoked by facial expressions differed across ROI pairs. Placebo Condition. For each facial expression, significant pairwise correlations were found for each ROI pair (Fig. 4). However, there were no significant differences among the functional connectivity maps (FCMs) for different facial expressions. OT Condition. Because the three-way interaction (treatment ×

expression × ROI pair) was significant, analyses were conducted for 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 interaction between expression and treatment for a given ROI pair are presented 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 the amygdala 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). As shown in Fig. 5B, significant interactions were mainly induced by the reduced functional coupling (coded by warm colors) among these areas in the signal evoked by fearful faces after OT administration (fearful faces in the placebo condition vs. in the OT condition: 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 condition) vs. (aggressive vs. neutral in the OT condition)] were also found between the amygdala and areas in the occipital and inferior 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 between subregions of the PFC (particularly VLPFC) and areas in the occipital 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 temporal cortex (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 for fearful faces, these interactions were mainly induced by the reduced functional coupling (coded by warm colors in Fig. 5C) among these areas in the signal evoked by aggressive faces after OT 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 present for one ROI pair (TEO–DLPFC: P = 0.040), which was caused by the reduced functional coupling in the signal evoked by appeasing faces after OT administration (P = 0.003); no systemic pattern for appeasing faces was found similar to the ones observed for fearful and aggressive faces (Fig. 5D). This result indicates that OT administration had less of an effect on functional coupling between face-responsive ROIs in the signal evoked by appeasing faces compared with those evoked by fearful and aggressive faces. Discussion In the present study, we investigated the effects of OT on fMRI responses evoked by different facial expressions (neutral, fearful, aggressive, and appeasing) in face-responsive ROIs within the occipital cortex, inferior temporal cortex, area LIP of parietal E3126 | www.pnas.org/cgi/doi/10.1073/pnas.1508097112

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

cortex, PFC, and amygdala of monkeys. We found that OT did not alter the fMRI response to neutral faces; OT differentially altered responses to emotional faces, significantly reducing responses to both fearful and aggressive faces while not changing the response to appeasing faces; and OT reduced functional coupling between the amygdala and areas in the occipital and inferior temporal cortex selectively in response to fearful and aggressive faces, but not neutral or appeasing faces. Here, we discuss the significance of these findings to understand the underlying neural mechanisms of the effects of OT. OT Modulates Brain Activity in Multiple Neural Systems. In the present study, in addition to the amygdala, we selected ROIs in the ventral visual pathway (from V1 to area TE), which respond selectively to visual features relevant for object identification, such as color, shape, and texture (27). We also investigated the effects of OT on brain activity in FEF and LIP, which are heavily interconnected and important for visual attention (28). Finally, we examined OT’s modulatory effects on three subregions of the PFC: DLPFC, VLPFC, and OFC (29), which are areas known to play important roles in emotion-related cognitive behavior. For example, OFC damage is associated with deficits in facial expression recognition (30); VLPFC is sensitive to changes in facial features, expressions, and gaze direction (31); and DLPFC is thought to play a central regulatory role in emotion processing (32). Similar modulatory effects of OT were found in all these ROIs: OT differentially altered responses to emotional faces. Thus, we provide the first evidence in monkeys, to our knowledge, that intranasally administered OT modulates brain activity in multiple neural systems, including those mediating visual perception (the occipital and inferior temporal cortex), emotion (the amygdala), attention (LIP and FEF), and higher cognitive function (PFC). Our findings are consistent with human neuroimaging results. Kirsch et al. found that OT reduces responses to Liu et al.

OT Reduces Brain Activity Evoked by Negative Emotional Stimuli.

Here, we demonstrate that intranasally administered OT in monkeys reduces activity in face-responsive ROIs to fearful and aggressive faces, but not to neutral or appeasing faces. Consistent with our findings, a previous study found that intranasally administered OT reduced monkeys’ attention to negative facial expressions (fear grin and open-mouth threat), but not to neutral or nonsocial stimuli (24). Although our results showed robust effects of OT on fearful and aggressive faces, they also showed that OT did not significantly alter the valence effect elicited by appeasing faces in the placebo condition. The appeasing faces we used, those expressing lip smack, could be considered an affiliative expression (33). It is possible that the lack of effect seen for appeasing faces in our study relates to the inconsistent results reported in humans during the viewing of happy faces (another affiliative expression) Liu et al.

How Does OT Modulate the Face-Responsive Network? In the present study, we found that the amygdala and many face-responsive cortical areas, including the occipital cortex, inferior temporal cortex, parietal cortex, and PFC, exhibited similar patterns of modulation after OT administration. These modulatory effects could 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 monkey brain, OT receptors are most robustly expressed in the nucleus basalis of Meynert, the superficial gray layer of the superior colliculus, the pedunculopontine tegmental nucleus, the trapezoid body, and the ventromedial hypothalamus (37). Although (a high density of) OT receptors have not been found in the selected ROIs of the present study, including the amygdala, activity in these ROIs may be modulated by projections from areas that contain OT receptors, and thereby influence social behavior and cognition. For example, the nucleus basalis of Meynert provides heavy cholinergic inputs to both the basolateral amygdala and cerebral 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 some social behavior and cognition (e.g., social communication) are mediated by AVP1A receptors (41, 42). AVP1A receptors are densely located in the prefrontal, cingulate, pyriform, and entorhinal cortex, as well as the presubiculum and mammillary bodies, but are also found in the amygdala, bed nucleus of the stria terminalis, lateral septum, hypothalamus, and brainstem (43). Two of these areas were selected as ROIs in the present study, the amygdala and PFC, which exhibited a pattern of PNAS | Published online May 26, 2015 | E3127

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fear-inducing stimuli in the amygdala (15). Domes et al. also found that OT attenuates activation in the amygdala, as well as in frontal and temporal areas, for angry and fearful faces (16). Furthermore, our findings may help explain OT’s modulatory effects on social behavior and cognition in monkeys. For example, a recent study in monkeys found that intranasal OT mitigates the influence of emotional faces on attention (23), which may reflect the effect of OT on brain structures involved in emotion (the amygdala) and attention (FEF and LIP) found in the present study. Our findings demonstrate homologies between monkeys and humans in the neural circuits mediating the effects of OT and provide key information for understanding the behavioral consequences of OT administration in monkeys.

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

after OT administration. For instance, Domes et al. reported that OT reduces the amygdala fMRI activation evoked by happy faces (16), whereas another study found enhanced responses to happy faces after OT administration (3). The discrepancy between our results and human findings may also be rooted in differences between the meaning of lip smack in macaques and a happy 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 the neural response to an aggressive facial expression (open-mouth threat) may depend on the animal’s rank in the social hierarchy. However, regardless of the difference between fearful and aggressive faces in the placebo condition, OT had a similar modulatory effect on responses to fearful and aggressive faces, which were both reduced. These distinct findings suggest that the neural mechanisms underlying the effect of OT on responses to fearful and aggressive faces may differ. In support of this idea, although we found that the effect of OT on responses to fearful and aggressive faces was similar across the different ROIs, OT administration differentially altered FCMs evoked by fearful and aggressive faces: reducing functional coupling between the PFC (especially VLPFC) and areas in the occipital and inferior temporal cortex in the signal evoked by aggressive faces, but not fearful 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 and aggressive) emotional stimuli. Importantly, our results demonstrate homologies between monkeys and humans not only in the neural circuits mediating the effects of OT but also in the underlying neural mechanisms of OT’s effects on social behavior and cognition. The results thus support the idea that the monkey is an ideal animal model to further explore the development of OT-based pharmacological strategies to treat patients with dysfunctional social behavior.

modulation after OT administration similar to the other selected ROIs. Neuroanatomical studies in monkeys have revealed that the amygdala and PFC are interconnected with the other selected ROIs (44–48). For example, feedback projections from the amygdala terminate in virtually all areas in the inferior temporal and occipital cortex, including V1 (44). Therefore, the observed effects of OT in the present study could be mediated by AVP1A receptors in the amygdala and/or PFC. This is consistent with our findings that the other selected ROIs showed similar valence effects to those observed in the amygdala and PFC in the placebo condition (e.g., fear grin consistently elicited the greatest response), that the reductions of activity by OT were similar in all of the selected ROIs [e.g., OT selectively affected the processing of negative (fearful and aggressive) emotional stimuli], and that the functional coupling between the amygdala/PFC and inferior temporal/occipital cortex evoked by negative (fearful and aggressive) emotional faces was reduced after OT administration. Thus, the observed alterations in brain activity during the perception of facial expressions after OT administration may be mediated 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 behavior and cognition through interactions with both OT and AVP receptors. Future studies (e.g., local injection of OT into specific areas that contain OT receptors) are needed to further explore the neural mechanisms underlying OT’s modulatory effects. Taken together, our results suggest that activity evoked by emotional stimuli, especially negative stimuli, is systematically altered after OT administration within multiple neural systems, including those mediating visual perception (the occipital and inferior temporal cortex), emotion (the amygdala), attention (LIP and FEF), and higher cognitive function (PFC). In addition, our results demonstrate homologies between human and monkey in the neural circuits mediating the effects of OT, thereby supporting the use of the nonhuman primate as an animal model for further studies of OT’s effects. We anticipate that the results of such studies will deepen our understanding of how this neuropeptide affects the brain and, eventually, facilitate human clinical applications.

interleaved gradient-recalled echo planar imaging. Imaging parameters were 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; flip angle: 90°. A low-resolution anatomical scan was also acquired in the same session to serve as an anatomical reference (modified driven equilibrium Fourier 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°). To facilitate cortical surface alignment, we acquired high-resolution T1-weighted whole-brain anatomical scans in separate sessions, using the modified driven equilibrium 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 Procedures

Intranasal OT Administration. Intranasal administration has been widely used to examine the effects of OT on social behavior and cognition. Several previous studies have studied which OT delivery method elevates cerebrospinal fluid OT concentrations in rhesus macaques (20, 35, 36). The method used in the present study has been demonstrated to be effective in causing elevations of OT concentration in cerebrospinal fluid (35). Before beginning the experiment, the monkeys were habituated to receiving saline intranasal spray using atomizers (Intranasal Mucosal Atomization Device; Wolfe Tory Medical) attached to syringes (35). The animals’ heads were fixed during this procedure to minimize movement and enhance the reliability of dosing. This habituation procedure was repeated until the monkeys were completely relaxed during the nasal spray administration. On the day of the experiment, monkeys were transported in a primate chair 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 found to 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. A 40-min delay between drug administration and the start of scanning is similar to the timing used in previous monkey and human studies (20–22, 53), as elevations of OT concentration in cerebrospinal fluid have been found ∼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 three monkeys were scanned in two separate sessions per treatment (placebo or OT), resulting in a total of 30 runs (60 condition repetitions) per monkey per treatment. The order of treatments was randomized for each monkey, with at least 5 d intervening.

Subjects 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 acquired from the same primate breeding facility in the United States, where they had social group histories as well as group-housing experience until their transfer to the National Institute of Mental Health (NIMH) for quarantine at the age of approximately 4 y. After that, they were individually caged with auditory and visual contact with other conspecifics in the same colony room, which accommodates about 20 rhesus monkeys. All three animals used in this study had been housed at NIMH for 4–5 y before this experiment. Thus, all three subjects have had extensive social experience, which made them familiar with perception and interpretation of facial cues in conspecifics. All procedures followed the Institute of Laboratory Animal Research (part of the National Research Council of the National Academy of Sciences) guidelines and were approved by the NIMH Animal Care and Use Committee. Each monkey was surgically implanted with a magnetic resonance (MR)-compatible head post under sterile conditions, using isoflurane anesthesia. After recovery, subjects were trained to sit in a plastic restraint chair and fixate a central target for long durations with their heads fixed, facing a screen on which visual stimuli were presented (49, 50). Brain Activity Measurements. Functional and anatomical MRI scanning was carried out in the Neurophysiology Imaging Facility Core [NIMH, National Institute of Neurological Disorders and Stroke (NINDS), National Eye Institute (NEI)]. Before each scanning session, an exogenous contrast agent [monocrystalline iron oxide nanocolloid (MION)] was injected into the femoral or external saphenous vein (12–15 mg/kg) to increase the contrast/noise ratio and to optimize the localization of fMRI signals (51, 52). Imaging data were collected 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

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Experimental Design and Task. All stimuli used in this experiment were identical to the ones used in Hadj-Bouziane et al. (34, 49). Briefly, the stimuli were color images of facial expressions displayed by eight unfamiliar macaque monkeys (frontal view): neutral, fearful (fear grin), aggressive (openmouth threat), and appeasing (lip smack). We presented these four different facial expressions to each monkey in a block design using Presentation software (version 12.2, www.neurobs.com). Stimuli spanned a visual angle of 11° (maximal horizontal and/or vertical extent) and were presented foveally for 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. Stimuli from each facial expression were presented in blocks of 32 s each, interleaved with 20-s fixation blocks (neutral gray background). Individual runs began and ended with a fixation block. Each categorical block was presented twice in each run in a pseudorandom order. Different pseudorandom sequences were used in each run. To locate the ROIs, we also performed an independent functional localizer experiment in all three animals, using the same fMRI parameters as in the main experiment, but with a different set of stimuli. In the localizer experiment, the stimuli were presented in a block design. Grayscale photos of neutral monkey faces, Fourier-phase scrambled faces, familiar places, and familiar 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-s fixation blocks (neutral gray background). Individual runs began and ended with 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 a total of 15–16 runs per monkey. In both the facial expression and localizer experiments, the monkeys were required to maintain fixation on a square superimposed on the stimuli to receive a liquid reward. In the reward schedule, the frequency of reward increased as the duration of fixation increased (49, 50). Eye position was monitored with an infrared pupil tracking system (iView, Inc).

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

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face-responsive ROIs. These regions did not show significant differences in response properties in both placebo and OT condition, relative to ROIs that included them. Responses to neutral and emotional faces. The signal in the facial expression experiment was extracted from face-responsive ROIs. We then calculated the response to each facial expression within each run (averaged across two repetitions) and performed ANOVAs with repeated measures for two withinsubject factors (ROI and expression) and one between-subject factor (treatment), followed by post hoc analyses and tests for interactions. P values were Bonferroni corrected for the number of comparisons. For all of the ANOVAs, we included the monkey as the nuisance between-subject factor. This procedure allowed us to test for our factors of interest (described earlier) while also statistically controlling for nuisance variability among monkeys. Functional connectivity. To investigate the context-specific changes in functional connectivity, the median activity within each face-responsive ROI was extracted for each condition. The points selected for each condition were based on the MION response model (i.e., convolving the MION kernel with a square wave of 32 s, then scaled to have a magnitude of 1). To reduce the noise level of these time series, only points in which the amplitude of the response model was greater than 0.1 were included for each condition (i.e., 30 of 32 points were selected). Then a pairwise correlation was calculated between each pair of ROIs. To rule out correlations caused by the shared task input and motion, the response model and movement parameters were controlled (i.e., via partial correlations). The partial correlation coefficients were calculated for each facial expression within each run (concatenating the two repetitions). We then applied Fisher’s z transformation to these correlation coefficients to convert them into normally distributed variables suitable for parametric statistical testing. Similar ANOVAs to those for responses to neutral and emotional faces were conducted: two within-subject factors (ROI pair and expression) and one between-subject factor (treatment), followed by multiple post hoc analyses and tests for interactions. P values were Bonferroni corrected for the number of comparisons. We also included the monkey as a nuisance between-subject factor. Note that the values shown in Figs. 4 and 5 depict the raw, untransformed correlation values, which were not included in any statistical analysis. As previously reported, there were no systematic significant differences between hemispheres in the response profile of any of the three monkeys (34). Thus, the analyses shown in the Results were based on pooled data from both hemispheres. Moreover, results from the individual monkeys were similar. Thus, we present results averaged across all three monkeys.

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15. Kirsch P, et al. (2005) Oxytocin modulates neural circuitry for social cognition and fear in humans. J Neurosci 25(49):11489–11493. 16. Domes G, et al. (2007) Oxytocin attenuates amygdala responses to emotional faces regardless of valence. Biol Psychiatry 62(10):1187–1190. 17. Petrovic P, Kalisch R, Singer T, Dolan RJ (2008) Oxytocin attenuates affective evaluations of conditioned faces and amygdala activity. J Neurosci 28(26):6607–6615. 18. Lischke A, et al. (2012) Oxytocin increases amygdala reactivity to threatening scenes in females. Psychoneuroendocrinology 37(9):1431–1438. 19. Watson KK, Platt ML (2012) Of mice and monkeys: Using non-human primate models to bridge mouse- and human-based investigations of autism spectrum disorders. J Neurodev Disord 4(1):21. 20. Chang SW, Barter JW, Ebitz RB, Watson KK, Platt ML (2012) Inhaled oxytocin amplifies both vicarious reinforcement and self reinforcement in rhesus macaques (Macaca mulatta). Proc Natl Acad Sci USA 109(3):959–964. 21. Dal Monte O, Noble PL, Costa VD, Averbeck BB (2014) Oxytocin enhances attention to the eye region in rhesus monkeys. Front Neurosci 8:41. 22. Ebitz RB, Watson KK, Platt ML (2013) Oxytocin blunts social vigilance in the rhesus macaque. Proc Natl Acad Sci USA 110(28):11630–11635. 23. Landman R, Sharma J, Sur M, Desimone R (2014) Effect of distracting faces on visual selective attention in the monkey. Proc Natl Acad Sci USA 111(50):18037–18042. 24. Parr LA, Modi M, Siebert E, Young LJ (2013) Intranasal oxytocin selectively attenuates rhesus monkeys’ attention to negative facial expressions. Psychoneuroendocrinology 38(9):1748–1756. 25. Simpson EA, et al. (2014) Inhaled oxytocin increases positive social behaviors in newborn macaques. Proc Natl Acad Sci USA 111(19):6922–6927. 26. Parr LA (September 23, 2014) Intranasal oxytocin enhances socially-reinforced learning in rhesus monkeys. Front Behav Neurosci, 10.3389/fnbeh.2014.00278. 27. Ungerleider LG, Mishkin M (1982) Analysis of Visual Behavior (MIT Press, Cambridge, MA).

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across all sessions. Images were realigned to the mean volume of the localizer session. The data were smoothed with a 2-mm full-width halfmaximum Gaussian kernel. Signal intensity was normalized to the mean signal value within each run. For each voxel in both experiments, we performed a single univariate linear model fit to estimate the response amplitude for each condition. The model included a hemodynamic response predictor for each condition and regressors of no interest [baseline, movement parameters from realignment corrections, and signal drifts (linear as well as quadratic)]. A general linear model and a MION kernel were used to model the hemodynamic response function (56). Definition of face-responsive ROIs. All ROIs were defined based on the localizer experiment. For each monkey, face-responsive voxels corresponded to voxels significantly more active for neutral faces compared with a blank screen (P < 10−4 uncorrected). Therein, we identified the following anatomically defined areas as described in the Saleem and Logothetis stereotaxic atlas (57): the occipital cortex: V1, V2, V3, and V4; the inferior temporal cortex: the posterior portion (area TEO) and the anterior portion (area TE); the amygdala; the LIP; and the PFC: FEF, DLPFC, VLPFC, and OFC. V1 included the posterior half of operculum and the ascending and descending limbs of the calcarine sulcus, as well the most posterior part of the stem of this sulcus. We defined V2 on the ventral surface in the lip of the lower bank of the calcarine sulcus, including a small portion of the medial surface near the upper bank of this sulcus, and in islands of gray matter within the posterior portions of the lunate and parieto-occipital sulci. V3 was defined as a narrow strip of cortex, located immediately anterior to V2 and posterior to V4 both dorsally and ventrally in the hemisphere. V4 extended between the lunate sulcus and the sts on the prelunate gyrus and extending ventrally through the anterior bank of the inferior occipital sulcus, as previously described (58). We defined TEO as extending from just anterior to the inferior occipital sulcus rostrally for 1 cm, dorsally to include the fundus and ventral bank of the sts, and ventromedially to include the lateral bank of the occipitotemporal sulcus TE was defined as adjacent and rostral to TEO, extending to the tip of the temporal pole, dorsally to include the fundus and ventral bank of the sts, and ventromedially to include the lateral bank of the occipitotemporal sulcus. LIP included both the dorsal and ventral subregions within the lateral bank of the inferior parietal sulcus (59). PFC extended rostrally from the fundus of the arcuate sulcus to include the lateral, medial, and orbital cortex. Within the PFC, we delineated DLPFC as extending from the fundus of the principal sulcus medially to the dorsal lip of the cingulate sulcus, VLPFC as extending from the fundus of the principal sulcus ventrally to 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 defined within the rostral bank of the genu of the arcuate sulcus, extending anteriorly over the sulcal lip. The extent of the amygdala was determined from high-resolution structural MRI scans. The other localizer comparisons (e.g., faces vs. objects) resulted in more circumscribed regions (e.g., face-selective regions) located within the selected

PNAS | Published online May 26, 2015 | E3129

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NEUROSCIENCE

ACKNOWLEDGMENTS. This work was supported by the National Institute of Mental 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 for technical assistance; Katalin M. Gothard for providing the original monkey facial expression stimuli; and Biying Xu for providing the exogenous contrast agent.

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