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A Spatiotemporal Profile of In Vivo Cerebral Blood Flow Changes Following Intranasal Oxytocin in Humans

Open AccessPublished:October 18, 2014DOI:https://doi.org/10.1016/j.biopsych.2014.10.005

      Abstract

      Background

      Animal and human studies highlight the role of oxytocin in social cognition and behavior and the potential of intranasal oxytocin (IN-OT) to treat social impairment in individuals with neuropsychiatric disorders such as autism. However, extensive efforts to evaluate the central actions and therapeutic efficacy of IN-OT may be marred by the absence of data regarding its temporal dynamics and sites of action in the living human brain.

      Methods

      In a placebo-controlled study, we used arterial spin labeling to measure IN-OT-induced changes in resting regional cerebral blood flow (rCBF) in 32 healthy men. Volunteers were blinded regarding the nature of the compound they received. The rCBF data were acquired 15 min before and up to 78 min after onset of treatment onset (40 IU of IN-OT or placebo). The data were analyzed using mass univariate and multivariate pattern recognition techniques.

      Results

      We obtained robust evidence delineating an oxytocinergic network comprising regions expected to express oxytocin receptors, based on histologic evidence, and including core regions of the brain circuitry underpinning social cognition and emotion processing. Pattern recognition on rCBF maps indicated that IN-OT-induced changes were sustained over the entire posttreatment observation interval (25–78 min) and consistent with a pharmacodynamic profile showing a peak response at 39–51 min.

      Conclusions

      Our study provides the first visualization and quantification of IN-OT-induced changes in rCBF in the living human brain unaffected by cognitive, affective, or social manipulations. Our findings can inform theoretical and mechanistic models regarding IN-OT effects on typical and atypical social behavior and guide future experiments (e.g., regarding the timing of experimental manipulations).

      Keywords

      Animal research has demonstrated that oxytocin (OT) plays a key role in the development and regulation of mammalian social behavior (
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      ). Second, task-based blood oxygen level–dependent (BOLD) functional magnetic resonance imaging (fMRI) studies cannot address this question because they identify relative changes between experimental and control conditions and are not sensitive to a single physiologic parameter (
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      We expected to observe increases in rCBF over mainly limbic areas previously identified to express OT receptors in human postmortem brains (
      • Loup F.
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      Localization of oxytocin binding sites in the human brainstem and upper spinal cord: An autoradiographic study.
      ,
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      Immunohistochemical localization of oxytocin receptors in human brain.
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      Neuromodulation by oxytocin and vasopressin.
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      ). We mapped the distribution of effects of IN-OT using conventional mass univariate voxel-by-voxel analysis, allowing inferences regarding local regions. In the absence of an a priori pharmacodynamic model, we used multivariate pattern recognition (PR) on rCBF maps (
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      • Haxby J.V.
      Beyond mind-reading: Multi-voxel pattern analysis of fMRI data.
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      • Chen Y.
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      • Wang Z.
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      • et al.
      Quantification of cerebral blood flow as biomarker of drug effect: Arterial spin labeling phMRI after a single dose of oral citalopram.
      ,
      • Doyle O.M.
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      • Brittain C.
      • O’Daly O.G.
      • Williams S.C.
      • et al.
      Quantifying the attenuation of the ketamine pharmacological magnetic resonance imaging response in humans: A validation using antipsychotic and glutamatergic agents.
      ,
      • Marquand A.F.
      • O’Daly O.G.
      • De Simoni S.
      • Alsop D.C.
      • Maguire R.P.
      • Williams S.C.
      • et al.
      Dissociable effects of methylphenidate, atomoxetine and placebo on regional cerebral blood flow in healthy volunteers at rest: A multi-class pattern recognition approach.
      ,
      • Doyle O.M.
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      ) as IN-OT does (
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      • Gimpl G.
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      ), PR offers increased sensitivity compared with conventional mass univariate approaches. The overall pattern of rCBF changes at each temporal interval can be reduced by PR into a single metric—the probability that an rCBF image belongs to a particular class (here, IN-OT or placebo). Using these predictive probabilities, we created pharmacodynamic profiles of changes in brain physiology following IN-OT or placebo.

      Methods and Materials

      Participants

      We recruited 32 healthy men (IN-OT group, n = 16, mean age (SD) = 24.23 (1.75) years; placebo group, n = 16, mean age = 25.78 (4.44) years; t30 = 1.30, p = .21) based on previous power analyses (
      • Murphy K.
      • Harris A.D.
      • Diukova A.
      • Evans C.J.
      • Lythgoe D.J.
      • Zelaya F.
      • et al.
      Pulsed arterial spin labeling perfusion imaging at 3 T: Estimating the number of subjects required in common designs of clinical trials.
      ). Participants were screened for psychiatric conditions using Symptom Checklist-90-Revised (
      • Derogatis L.R.
      • Savitz K.L.
      The SCL-90-R and the Brief Symptom Inventory (BSI) in primary care.
      ) and Beck Depression Inventory-II (
      • Beck A.T.
      • Steer R.A.
      • Ball R.
      • Ranieri W.
      Comparison of Beck Depression Inventories -IA and -II in psychiatric outpatients.
      ) questionnaires, did not take any prescribed drugs, tested negative on a urine screening test for drugs of abuse, and consumed <28 units of alcohol per week and <5 cigarettes per day. Both parents of participants were white European to reduce genetic background variability. Participants abstained from alcohol and heavy exercise for 24 hours and abstained from any beverage or food in the 2 hours before scanning in the morning. Participants gave written informed consent. King’s College London Research Ethics Committee (PNM/10/11-160) approved the study.

      Design, Materials, and Procedure

      We employed a single-blinded, placebo-controlled design with two independent study arms. Before taking part, all participants were informed they would receive a neuropeptide and remain blinded to its name and that they might receive placebo until the postsession debriefing; 50% received IN-OT, and 50% received placebo. We obtained two baseline cerebral blood flow (CBF) images before participants came out of the scanner to receive 40 IU of IN-OT (Syntocinon; Novartis, Basel, Switzerland) or placebo (same composition as Syntocinon except for OT). We used 40 IU, the highest clinically applicable safe dose administered to human volunteers [e.g., in 14% of studies until 2011 (
      • MacDonald E.
      • Dadds M.R.
      • Brennan J.L.
      • Williams K.
      • Levy F.
      • Cauchi A.J.
      A review of safety, side-effects and subjective reactions to intranasal oxytocin in human research.
      ) and still being used (
      • MacDonald K.
      • MacDonald T.M.
      • Brune M.
      • Lamb K.
      • Wilson M.P.
      • Golshan S.
      • et al.
      Oxytocin and psychotherapy: A pilot study of its physiological, behavioral and subjective effects in males with depression.
      )] to maximize power. Use of this dose also ensured comparability with the study of Born et al. (
      • Born J.
      • Lange T.
      • Kern W.
      • McGregor G.P.
      • Bickel U.
      • Fehm H.L.
      Sniffing neuropeptides: A transnasal approach to the human brain.
      ) on vasopressin in the CSF using 40 IU as the minimum dose.
      Participants self-administered one puff (4 IU) of IN-OT (or placebo) every 30 seconds, alternating between nostrils. The administration phase lasted approximately 9 minutes including a 3-minute rest at the end. Participants returned to the scanner for two anatomic scans followed by eight CBF images spanning 25–78 minutes from the onset (henceforth called postadministration scans) of nasal spray administration (Figure 1A). Participants were instructed to lie still and maintain their gaze on a centrally placed fixation cross during scanning. We assessed participants’ levels of alertness (anchors: alert-drowsy) and excitement (anchors: excited-calm) using visual analog scales before acquiring each CBF image. The subjective ratings of one participant from the IN-OT group were lost because of a technical issue.
      Figure thumbnail gr1
      Figure 1Experimental design, subjective ratings, and global cerebral blood flow values (IN-OT group, n = 16; placebo group, n = 16). (A) Experimental design. (B) Οverall, participants’ levels of alertness (IN-OT, z = 2.57, p = .010; placebo, z = 2.20, p = .028) and excitement (IN-OT, z = 2.12, p = .034; placebo, z = 1.40, p = .16) linearly decreased over time. For VAS ratings, we measured the distance of the cursor from one extreme and converted to a score ranging from −50 (alert/excited) to 50 (drowsy/calm), with 0 being the midpoint. (C) Global cerebral blood flow values also linearly decreased over time (IN-OT, z = −2.82, p = .005; placebo, z = −2.45, p = .014). Error bars represent SE. Corrected p values reported. BSL, baseline; IN-OT, intranasal oxytocin; MRI, magnetic resonance imaging; rCBF, regional cerebral blood flow; VAS, visual analog scale.

      Image Acquisition and Preprocessing

      Images were acquired using a Signa HDx 3.0T magnetic resonance imaging scanner (General Electric, Milwaukee, Wisconsin). We employed the pulsed-continuous arterial spin labeling methodology (
      • Dai W.
      • Garcia D.
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      • Alsop D.C.
      Continuous flow-driven inversion for arterial spin labeling using pulsed radio frequency and gradient fields.
      ). The CBF maps (in standard physiologic units—mL blood/100 g tissue/min) were computed with a spatial resolution of 1 mm × 1 mm × 3 mm. Total acquisition time for each CBF map was 5.5 minutes. We also acquired a T2-weighted fast spin echo high spatial resolution structural image for coregistration and normalization purposes.
      We performed the following preprocessing steps (detailed in the Supplement. 1) We removed extracerebral signal from each participant’s T2 volume and created a binary brain mask. 2) We coregistered each CBF image to the corresponding T2 volume for each participant, correcting for interscan movement. 3) We removed extracerebral signal from CBF images by multiplying them by the binary brain mask. 4) We normalized the T2 volume to the Montreal Neurological Institute 2-mm T2 template, applying the transformation matrix to the coregistered, brain-only CBF images. 5) We smoothed the CBF images using an 8-mm Gaussian kernel. We restricted the search volume to gray matter voxels only using an explicit mask of voxels with a >.20 probability of being gray matter.

      Global CBF Measures and Subjective Ratings

      We extracted global CBF values using MarsBaR (http://marsbar.sourceforge.net/). We conducted the following analyses (Supplement): First, we performed a nonparametric test for a linear change in global CBF signal and subjective ratings over time (
      • Cuzick J.
      A Wilcoxon-type test for trend.
      ). Second, we investigated the association between changes in global CBF signal and self-ratings of alertness and excitement over time. Finally, we tested for the effect of treatment on subjective ratings and global CBF signal (averaged over baseline and postadministration scans) with the Treatment (IN-OT, placebo; between-subjects factor) × Period (baseline, postadministration; within-subjects factor) term in a mixed 2 × 2 analysis of variance model implemented in Stata (version 13; StataCorp LP, College Station, Texas), correcting for data dependence (
      • Williams R.L.
      A note on robust variance estimation for cluster-correlated data.
      ) and multiple testing using the sequential Holm-Bonferroni correction procedure (
      • Holm S.
      A simple sequentially rejective multiple test procedure.
      ).

      Whole-Brain Univariate Analyses: Mapping the Spatial Profile of IN-OT-Induced Changes in rCBF

      We implemented an analysis of covariance design to control for baseline differences (
      • Vickers A.J.
      • Altman D.G.
      Statistics notes: Analysing controlled trials with baseline and follow up measurements.
      ) using a flexible factorial model in SPM8 software (http://www.fil.ion.ucl.ac.uk/spm/software/spm8/), specifying the factors Subjects, Treatment, and Period. We used the Treatment × Period interaction term and an F contrast to test for brain regions where IN-OT led to changes in rCBF regardless of direction in the 25–78 minutes after administration. We conducted cluster-level inferences at α = .05, using familywise error (FWE) correction for multiple comparisons from a voxel-level cluster-forming threshold of Z > 2.3 (
      • Worsley K.J.
      Statistical analysis of activation images.
      ). The required cluster size threshold for the F contrast at α = .05 was calculated to be k = 1089 voxels using Analysis of Functional NeuroImages 3dClustSim (http://afni.nimh.nih.gov/pub/dist/doc/program_help/3dClustSim.html). To understand the nature of the effect, we extracted and plotted the data from each of the identified clusters, adjusting for the Treatment × Period contrast. To enhance the contrast between rCBF at baseline and after administration, we included global CBF values as nuisance covariates in the general linear model.

      PR: Investigating Temporal Dynamics of the Spatial Pattern of IN-OT-Induced Changes in rCBF

      We restricted PR analyses to an a priori–defined mask that included brain regions likely to contain oxytocin receptors, based on previous postmortem human brain studies (Supplement). Briefly, PR involves learning a pattern (a “model”) of brain voxels that can distinguish rCBF images as being acquired before or after treatment (our two “classes”). This trained model could be used to assign a label to a new, previously unseen image (“classification”). This procedure is repeated N times, each time using N-1 participants to learn the pattern (“training the model”) and applying it to images from the Nth (“left-out”) participant (“leave-one-out cross-validation” procedure). We used Gaussian process classification (GPC) (
      • Doyle O.M.
      • De Simoni S.
      • Schwarz A.J.
      • Brittain C.
      • O’Daly O.G.
      • Williams S.C.
      • et al.
      Quantifying the attenuation of the ketamine pharmacological magnetic resonance imaging response in humans: A validation using antipsychotic and glutamatergic agents.
      ,
      • Doyle O.M.
      • Ashburner J.
      • Zelaya F.O.
      • Williams S.C.
      • Mehta M.A.
      • Marquand A.F.
      Multivariate decoding of brain images using ordinal regression.
      ,
      • Rasmussen C.E.
      • Williams C.K.I.
      Gaussian Processes for Machine Learning.
      ) to estimate the probability that a previously unseen image from the Nth participant belongs to the posttreatment class (the “predictive probability”) (see the Supplement for more information on the GPC). A predictive probability >.5 was used to assign an image to the posttreatment class. The statistical significance of the performance of the model was estimated using permutations (i.e., repeating the above-described procedure after randomly mixing the training image labels 1000 times to test the null hypothesis that the performance is not greater than chance [50%]—of no predictive value). Figure 2 details a schematic representation of GPC analysis. We averaged pairs of consecutive scans to improve the signal-to-noise ratio, potentially improving classification accuracy (
      • Marquand A.F.
      • O’Daly O.G.
      • De Simoni S.
      • Alsop D.C.
      • Maguire R.P.
      • Williams S.C.
      • et al.
      Dissociable effects of methylphenidate, atomoxetine and placebo on regional cerebral blood flow in healthy volunteers at rest: A multi-class pattern recognition approach.
      ).
      Figure thumbnail gr2
      Figure 2Schematic analysis pipeline. (A) Experimental setup. Indicative rCBF maps are presented for illustration purposes. (B) Training and testing the Gaussian process classifier model on each participant and an illustration of the baseline–post nasal spray continuum. rCBF, regional cerebral blood flow.
      We used the predictive probabilities as the main outcome measure and as a proxy to create a pharmacodynamic profile of the IN-OT perfusion effect. We compared scans after administration with baseline for each study arm separately because the administration of IN-OT and placebo to different individuals was expected to inflate the between-group variance, obscuring the sensitivity of GPC to the expected IN-OT effect. Cross-group multivariate classification was not part of our design (see Supplemental Table S1 for results). However, to obtain a formal measure of cross-group classification performance, we implemented a mixed Treatment × Period (using the seven postadministration subperiods defined by each possible pair of consecutive rCBF images) analysis of variance on predictive probabilities. Finally, we constructed maps to visualize the distributed discriminative spatial pattern of multivariate weights driving the classification.

      Results

      Global CBF Measures and Subjective Ratings

      We observed a general linear decrease over time in participants’ levels of alertness and excitement and in global CBF values in both groups (Figure 1B, C and Supplemental Results). Self-reported levels of alertness and excitement correlated with global CBF values (alertness, r = .16, p = .009; excitement, r = .23, p < .001). This association and the significant global decrease in rCBF over time, which might prevent the identification of significant changes in CBF in small regions, provided a suitable rationale for including global CBF as a confounding covariate in the analyses. We did not observe Treatment or Period effects on global CBF values (Supplemental Results and Supplemental Table S2).

      Univariate Analyses: Mapping Spatial Profile of IN-OT-Induced Changes in rCBF

      The F contrast identified four clusters showing a significant Treatment × Period interaction in rCBF in the 25–78 minute postadministration interval (Table 1 and Figure 3). Clusters extended over a network of regions including 1) left hemisphere limbic and midbrain/brainstem regions, including amygdala, hippocampus, caudate nucleus, ventral striatum and pallidum, septal and hypothalamic nuclei, substantia nigra, and pontine brainstem nuclei; 2) bilateral dorsal anterior and middle cingulate cortices; 3) inferior frontal gyrus, anterior insula, frontal and parietal opercula, and superior temporal gyrus, extending to the plana temporale and polare and the supramarginal gyrus in inferior parietal cortex; and 4) right hemisphere cerebellum. Directional T contrasts for the interaction effect identified the same clusters (clusters 1 and 3, pFWE < .001, k = 7360; cluster 2, pFWE = .019, k = 2114; cluster 4, pFWE = .041, k = 1767). We extracted and plotted data from each of these clusters, observing a crossover interaction pattern where the administration of IN-OT increased rCBF in clusters 1–3 (compared with baseline), whereas the reverse pattern was observed for the placebo group. The opposite pattern was observed in the cerebellum (Figure 3).
      Table 1Clusters Showing a Significant Treatment × Period Interaction in rCBF in the 25–78 minute Postadministration Interval (F Contrast)
      Peak Coordinates
      Cluster DescriptionHemispherekpxyzDescription
      Cluster 1
       Caudate nucleus, ventral striatum, pallidum, amygdala, hippocampus, septal nuclei, hypothalamus, ventral midbrain (ventral tegmental area, substantia nigra), pontine tegmentumLeft1999<.05−14−30−36Dorsal midbrain
      −10−18−26Ventral midbrain
      −1042Pallidum
      Cluster 2
       Anterior and middle cingulate corticesBilateral1539<.05−21024Anterior cingulate cortex
      −2−1232Middle cingulate cortex
      Cluster 3
       Inferior frontal gyrus, anterior insula, planum polare, transverse temporal gyrus, planum temporale, superior temporal gyrus, inferior parietal cortex–supramarginal gyrus, frontal operculum, parietal operculumLeft2816<.05−500−4Superior temporal gyrus
      −58−220Middle temporal gyrus
      −58−246Superior temporal gyrus
      Cluster 4
       CerebellumRight1244<.0528−68−42Cerebellum (lobule VIIa, crus II)
      14−66−42Cerebellum (lobule VIIIa)
      44−56−50Cerebellum (lobule VIIa, crus I)
      The required cluster size threshold at α = .05 was calculated to be k = 1089 voxels, using the Analysis of Functional NeuroImages program 3dClustSim.
      rCBF, regional cerebral blood flow.
      Figure thumbnail gr3
      Figure 3Statistical parametric maps for the four clusters showing a significant Treatment (intranasal oxytocin [n = 16], placebo [n = 16]) × Period (baseline, after administration) interaction (F contrast) over the entire observation interval of 25–78 minutes following the onset of treatment. Inserted graphs plot the extracted (first eigenvariate), F contrast adjusted rCBF values for each cluster to illustrate the interaction effect. Error bars represent SE. The right-hand side of each image corresponds to the participant’s right side. Slice numbers indicate Montreal Neurological Institute coordinates. rCBF, regional cerebral blood flow.

      PR Analyses: Investigating Temporal Dynamics of IN-OT-Induced Changes in rCBF

      Classification Accuracies

      The classification accuracies for the post IN-OT class (compared with the baseline class) were >80% at all time intervals and significantly different from chance. For the post placebo class (compared with baseline), classification accuracies ranged from 38%–81% and did not differ significantly from chance except at the 32–44 minute interval (Table 2).
      Table 2Performance Parameters for the Gaussian Process Classification Model
      ContrastAccuracyp
      Holm-Bonferroni corrected values.
      Sensitivity (%)Specificity (%)Predictive p (Postnasal Spray | rCBF Map) (M ± SE)
      Post IN-OT Class
       25–38 min.83<.00181.2581.25.69 ± .05
       32–44 min.83<.00181.2581.25.73 ± .05
       39–51 min.94<.00193.7593.75.80 ± .03
       45–58 min1.00<.001100.00100.00.77 ± .03
       52–65 min.88<.00187.587.5.73 ± .04
       59–71 min.88<.00187.587.5.71 ± .05
       66–78 min.83<.00181.2581.25.70 ± .05
      Post Placebo Class
       25–38 min.69>.0568.7568.75.64 ± .05
       32–44 min.81<.00181.2581.25.66 ± .05
       39–51 min.63>.0562.562.5.62 ± .05
       45–58 min.69>.0568.7568.75.58 ± .05
       52–65 min.38>.0537.537.5.55 ± .05
       59–71 min.56>.0556.2556.25.59 ± .05
       66–78 min.63>.0562.562.5.61 ± .05
      Performance of the Gaussian process classification was assessed using the leave-one-out procedure; statistical significance of the classification accuracies was determined by random permutation and adjusted for multiple testing using the Holm-Bonferroni correction procedure.
      IN-OT, intranasal oxytocin; rCBF, regional cerebral blood flow.
      a Holm-Bonferroni corrected values.

      Predictive Probabilities

      Statistical analysis of cross-group classification performance using the predictive probabilities confirmed the above-described pattern. We observed a significant main effect for Treatment (but not Period) and a significant Treatment × Period interaction (Table 3, Figure 4A). Predictive probabilities were significantly higher for the post IN-OT class compared with post placebo class at 39–51 minute, 45–58 minute, and 52–65 minute intervals. Similarly, there was an effect of Period in the post IN-OT but not the post placebo group. Figure 4A shows that the averaged predictive probabilities for the post IN-OT class peaked at the 39–51 minutes post IN-OT interval, followed by a gradual diminution over time. Figure 4B shows the multivariate map of the discriminative spatial pattern underpinning classification at the 39–51 minute interval in the post IN-OT and placebo groups.
      Table 3Treatment × Period ANOVA on Predictive Probabilities Computed Using Gaussian Process Classification on rCBF Maps
      χ2
      The bootstrap procedure in Stata uses χ2 statistics to test for statistical significance, which we report (equivalent F values can be obtained by dividing the χ2 statistic by its degrees of freedom).
      df
      The bootstrap procedure in Stata uses χ2 statistics to test for statistical significance, which we report (equivalent F values can be obtained by dividing the χ2 statistic by its degrees of freedom).
      p
      Predictive Probabilities
       Treatment5.431.02
       Period7.346.29
       Treatment × Period13.596.035
      Simple Effects Analyses
      Reported p values are adjusted for multiple testing using the sequential Holm-Bonferroni correction procedure.
       Treatment effect at 25–38 min.491.49
       Treatment effect at 32–44 min.941.66
       Treatment effect at 39–51 min8.811.018
       Treatment effect at 45–58 min9.981.011
       Treatment effect at 52–65 min6.971.042
       Treatment effect at 59–71 min3.441.25
       Treatment effect at 66–78 min1.691.58
       Period effect in IN-OT group15.066.040
       Period effect in placebo group10.166.12
      ANOVA, analysis of variance; IN-OT, intranasal oxytocin; rCBF, regional cerebral blood flow.
      a The bootstrap procedure in Stata uses χ2 statistics to test for statistical significance, which we report (equivalent F values can be obtained by dividing the χ2 statistic by its degrees of freedom).
      b Reported p values are adjusted for multiple testing using the sequential Holm-Bonferroni correction procedure.
      Figure thumbnail gr4
      Figure 4Pattern recognition analyses. (A) Estimated marginal mean predictive probabilities for the post nasal spray administration class (y axis) (and 95% confidence intervals) for the Treatment (IN-OT [n = 16], placebo [n = 16]) × Period (baseline, after administration) interaction as a function of time interval, using an a priori defined mask of brain regions likely to express oxytocin receptors. Note the temporal overlap between adjacent time intervals; to sample the entire postoxytocin period, we averaged all pairs of adjacent regional cerebral blood flow maps. We observed a significant main effect for Treatment [χ21 = 5.43, p = .020], but not for Period [χ26 = 7.34, p = .29], and a significant Treatment × Period interaction [χ26 = 13.59, p = .035] (corrected p values reported). The mean predictive probabilities for the post–IN-OTclass followed a pharmacodynamic profile showing a peak response at 39–51 minutes, followed by a gradual diminution of effects. (B) Multivariate maps of normalized weight vectors from the Gaussian process classification (g-maps) at the 39–51 minute interval that contrast the post–IN-OT/placebo classes to the baseline class. Positive coefficients (red color scale) indicate a positive contribution to the prediction for each class, and negative coefficients (blue color scale) indicate a negative contribution. A positive g-map coefficient for a particular voxel indicates a higher overall β weight for the post–IN-OT/placebo class, and similarly a negative g-map coefficient indicates a higher overall β score for the baseline class. A region with a high weight cannot be interpreted as driving the classification; the whole pattern of weights drives the classification. These maps cannot be used to make inferences about local activation. The right-hand side of each image corresponds to the participant’s right side. Slice numbers indicate Montreal Neurological Institute coordinates. (C) The a priori defined mask of brain regions likely to express oxytocin receptors included the subcallosal area (including basal forebrain regions), nucleus accumbens, caudate nucleus, putamen, globus pallidus, amygdala, hippocampus, thalamus, and hypothalamus. The hypothalamus was defined with a sphere centered on Montreal Neurological Institute coordinates (x y z: 0, −4, −8) using a 12-mm radius (
      • Baroncini M.
      • Jissendi P.
      • Balland E.
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      ). The remaining regions of interest were defined using the Harvard-Oxford cortical and subcortical structural atlases in FSLview (http://fsl.fmrib.ox.ac.uk). CI, confidence interval; IN-OT, intranasal oxytocin; rCBF, regional cerebral blood flow.

      Discussion

      Using arterial spin labeling as a pharmacodynamic biomarker (
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      Quantifying the attenuation of the ketamine pharmacological magnetic resonance imaging response in humans: A validation using antipsychotic and glutamatergic agents.
      ,
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      • Maguire R.P.
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      Dissociable effects of methylphenidate, atomoxetine and placebo on regional cerebral blood flow in healthy volunteers at rest: A multi-class pattern recognition approach.
      ,
      • Viviani R.
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      ), we visualized and quantified, for the first time in living human brain, IN-OT-induced changes in rCBF unaffected by concomitant cognitive, affective, or social manipulations. Confirming predictions from postmortem histologic studies (
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      Localization of high-affinity binding sites for oxytocin and vasopressin in the human brain. An autoradiographic study.
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      Immunohistochemical localization of oxytocin receptors in human brain.
      ), we delineated an oxytocinergic network comprising regions expected to express OT receptors and that are involved in social cognition and emotion processing (
      • Adolphs R.
      The neurobiology of social cognition.
      ,
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      ). Addressing the lack of a temporal dynamics model for the human brain, GPC indicated that IN-OT-induced changes in rCBF were sustained over the posttreatment observation interval of 25–78 minutes after the onset of IN-OT administration and were consistent with a pharmacodynamic profile showing a peak response at 39–51 minutes, followed by a gradual diminution of effects.

      Mapping the Oxytocinergic Network in the Human Brain

      The significant Treatment × Period crossover interaction identified cortical and subcortical regions showing higher rCBF after treatment in the IN-OT group compared with the placebo group, controlling for baseline differences. Most of the subcortical limbic areas and the anterior cingulate gyrus have been previously reported to express OT receptors in postmortem human brains (
      • Loup F.
      • Tribollet E.
      • Dubois-Dauphin M.
      • Dreifuss J.J.
      Localization of high-affinity binding sites for oxytocin and vasopressin in the human brain. An autoradiographic study.
      ,
      • Loup F.
      • Tribollet E.
      • Dubois-Dauphin M.
      • Pizzolato G.
      • Dreifuss J.J.
      Localization of oxytocin binding sites in the human brainstem and upper spinal cord: An autoradiographic study.
      ,
      • Boccia M.L.
      • Petrusz P.
      • Suzuki K.
      • Marson L.
      • Pedersen C.A.
      Immunohistochemical localization of oxytocin receptors in human brain.
      ). In the rat, OT receptors were also identified in insular and temporal lobe regions (
      • Gimpl G.
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      • Phelps S.M.
      Oxytocin receptor density is associated with male mating tactics and social monogamy.
      ), but no evidence exists for humans.
      The brain areas showing increased rCBF following IN-OT at rest are part of a distributed “social brain” network (
      • Adolphs R.
      The neurobiology of social cognition.
      ,
      • Adolphs R.
      The social brain: Neural basis of social knowledge.
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      • Amodio D.M.
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      Is there a core neural network in empathy? An fMRI based quantitative meta-analysis.
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      • Carr L.
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      Intranasal administration of oxytocin increases envy and schadenfreude (gloating).
      ) (see the Supplement for a discussion of findings in relation to the role of OT in centrally mediated physiologic functions). This network underpins the processing of social and emotional stimuli and the expression of social and affiliative behavior (
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      • Brammer M.J.
      • et al.
      Exploring the social brain in schizophrenia: Left prefrontal underactivation during mental state attribution.
      ), those involved in cognitive and emotional empathy (
      • Fan Y.
      • Duncan N.W.
      • de Greck M.
      • Northoff G.
      Is there a core neural network in empathy? An fMRI based quantitative meta-analysis.
      ,
      • Carr L.
      • Iacoboni M.
      • Dubeau M.C.
      • Mazziotta J.C.
      • Lenzi G.L.
      Neural mechanisms of empathy in humans: A relay from neural systems for imitation to limbic areas.
      ,
      • Iacoboni M.
      Imitation, empathy, and mirror neurons.
      ,
      • Shamay-Tsoory S.G.
      • Aharon-Peretz J.
      • Perry D.
      Two systems for empathy: A double dissociation between emotional and cognitive empathy in inferior frontal gyrus versus ventromedial prefrontal lesions.
      ,
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      • Fischer M.
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      • Harari H.
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      Intranasal administration of oxytocin increases envy and schadenfreude (gloating).
      ) or theory of mind and mentalizing (
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      • Labuschagne I.
      • Phan K.L.
      • Wood A.
      • Angstadt M.
      • Chua P.
      • Heinrichs M.
      • et al.
      Medial frontal hyperactivity to sad faces in generalized social anxiety disorder and modulation by oxytocin.
      ,
      • Lischke A.
      • Berger C.
      • Prehn K.
      • Heinrichs M.
      • Herpertz S.C.
      • Domes G.
      Intranasal oxytocin enhances emotion recognition from dynamic facial expressions and leaves eye-gaze unaffected.
      ,
      • Lischke A.
      • Gamer M.
      • Berger C.
      • Grossmann A.
      • Hauenstein K.
      • Heinrichs M.
      • et al.
      Oxytocin increases amygdala reactivity to threatening scenes in females.
      ,
      • Petrovic P.
      • Kalisch R.
      • Singer T.
      • Dolan R.J.
      Oxytocin attenuates affective evaluations of conditioned faces and amygdala activity.
      ,
      • Pincus D.
      • Kose S.
      • Arana A.
      • Johnson K.
      • Morgan P.S.
      • Borckardt J.
      • et al.
      Inverse effects of oxytocin on attributing mental activity to others in depressed and healthy subjects: A double-blind placebo controlled FMRI study.
      ,
      • Riem M.M.
      • Bakermans-Kranenburg M.J.
      • Pieper S.
      • Tops M.
      • Boksem M.A.
      • Vermeiren R.R.
      • et al.
      Oxytocin modulates amygdala, insula, and inferior frontal gyrus responses to infant crying: A randomized controlled trial.
      ,
      • Riem M.M.
      • van I.M.H.
      • Tops M.
      • Boksem M.A.
      • Rombouts S.A.
      • Bakermans-Kranenburg M.J.
      No laughing matter: Intranasal oxytocin administration changes functional brain connectivity during exposure to infant laughter.
      ,
      • Schulze L.
      • Lischke A.
      • Greif J.
      • Herpertz S.C.
      • Heinrichs M.
      • Domes G.
      Oxytocin increases recognition of masked emotional faces.
      ) and has been linked to differences in peripheral OT levels (
      • Atzil S.
      • Hendler T.
      • Zagoory-Sharon O.
      • Winetraub Y.
      • Feldman R.
      Synchrony and specificity in the maternal and the paternal brain: Relations to oxytocin and vasopressin.
      ). A meta-analysis of studies involving the processing of emotional stimuli showed that IN-OT increased BOLD signal over a single cluster centered on the left insula, extending into the superior temporal and paracentral gyri (
      • Rocchetti M.
      • Radua J.
      • Paloyelis Y.
      • Xenaki L.A.
      • Frascarelli M.
      • Caverzasi E.
      • et al.
      Neurofunctional maps of the “maternal brain” and the effects of oxytocin: A multimodal voxel-based meta-analysis.
      ). Additionally, IN-OT enhanced neural activity within the oxytocinergic network in children with autism spectrum disorder while making social judgments (
      • Gordon I.
      • Vander Wyk B.C.
      • Bennett R.H.
      • Cordeaux C.
      • Lucas M.V.
      • Eilbott J.A.
      • et al.
      Oxytocin enhances brain function in children with autism.
      ). A single IN-OT dose (compared with placebo) also modulates functional connectivity between nodes of the oxytocinergic network when participants engage in tasks requiring social cognitive or emotional processing (
      • Meyer-Lindenberg A.
      • Domes G.
      • Kirsch P.
      • Heinrichs M.
      Oxytocin and vasopressin in the human brain: Social neuropeptides for translational medicine.
      ,
      • Baumgartner T.
      • Heinrichs M.
      • Vonlanthen A.
      • Fischbacher U.
      • Fehr E.
      Oxytocin shapes the neural circuitry of trust and trust adaptation in humans.
      ,
      • Domes G.
      • Heinrichs M.
      • Michel A.
      • Berger C.
      • Herpertz S.C.
      Oxytocin improves “mind-reading” in humans.
      ,
      • Domes G.
      • Lischke A.
      • Berger C.
      • Grossmann A.
      • Hauenstein K.
      • Heinrichs M.
      • et al.
      Effects of intranasal oxytocin on emotional face processing in women.
      ,
      • Gamer M.
      Does the amygdala mediate oxytocin effects on socially reinforced learning?.
      ,
      • Labuschagne I.
      • Phan K.L.
      • Wood A.
      • Angstadt M.
      • Chua P.
      • Heinrichs M.
      • et al.
      Oxytocin attenuates amygdala reactivity to fear in generalized social anxiety disorder.
      ,
      • Labuschagne I.
      • Phan K.L.
      • Wood A.
      • Angstadt M.
      • Chua P.
      • Heinrichs M.
      • et al.
      Medial frontal hyperactivity to sad faces in generalized social anxiety disorder and modulation by oxytocin.
      ,
      • Lischke A.
      • Berger C.
      • Prehn K.
      • Heinrichs M.
      • Herpertz S.C.
      • Domes G.
      Intranasal oxytocin enhances emotion recognition from dynamic facial expressions and leaves eye-gaze unaffected.
      ,
      • Lischke A.
      • Gamer M.
      • Berger C.
      • Grossmann A.
      • Hauenstein K.
      • Heinrichs M.
      • et al.
      Oxytocin increases amygdala reactivity to threatening scenes in females.
      ,
      • Petrovic P.
      • Kalisch R.
      • Singer T.
      • Dolan R.J.
      Oxytocin attenuates affective evaluations of conditioned faces and amygdala activity.
      ,
      • Pincus D.
      • Kose S.
      • Arana A.
      • Johnson K.
      • Morgan P.S.
      • Borckardt J.
      • et al.
      Inverse effects of oxytocin on attributing mental activity to others in depressed and healthy subjects: A double-blind placebo controlled FMRI study.
      ,
      • Riem M.M.
      • Bakermans-Kranenburg M.J.
      • Pieper S.
      • Tops M.
      • Boksem M.A.
      • Vermeiren R.R.
      • et al.
      Oxytocin modulates amygdala, insula, and inferior frontal gyrus responses to infant crying: A randomized controlled trial.
      ,
      • Riem M.M.
      • van I.M.H.
      • Tops M.
      • Boksem M.A.
      • Rombouts S.A.
      • Bakermans-Kranenburg M.J.
      No laughing matter: Intranasal oxytocin administration changes functional brain connectivity during exposure to infant laughter.
      ,
      • Schulze L.
      • Lischke A.
      • Greif J.
      • Herpertz S.C.
      • Heinrichs M.
      • Domes G.
      Oxytocin increases recognition of masked emotional faces.
      ) or are at rest (
      • Sripada C.S.
      • Phan K.L.
      • Labuschagne I.
      • Welsh R.
      • Nathan P.J.
      • Wood A.G.
      Oxytocin enhances resting-state connectivity between amygdala and medial frontal cortex.
      ). For example, Striepens et al. (
      • Striepens N.
      • Scheele D.
      • Kendrick K.M.
      • Becker B.
      • Schafer L.
      • Schwalba K.
      • et al.
      Oxytocin facilitates protective responses to aversive social stimuli in males.
      ) reported that IN-OT (compared with placebo) enhanced functional connectivity among the left amygdala, left anterior insula, and left inferior frontal gyrus, whereas Riem et al. (
      • Riem M.M.
      • van Ijzendoorn M.H.
      • Tops M.
      • Boksem M.A.
      • Rombouts S.A.
      • Bakermans-Kranenburg M.J.
      Oxytocin effects on complex brain networks are moderated by experiences of maternal love withdrawal.
      ) reported increased functional connectivity among the cingulate and somatosensory cortices and the cerebellum.
      The reverse pattern was shown by rCBF changes in the right posterior cerebellar lobules: rCBF decreased after IN-OT treatment compared with placebo. These cerebellar lobules show strong functional connections with cerebral limbic association networks and networks related to executive control (
      • Buckner R.L.
      The cerebellum and cognitive function: 25 years of insight from anatomy and neuroimaging.
      ). This right laterality is consistent with the predominantly contralateral cerebral-cerebellar mappings (
      • Buckner R.L.
      The cerebellum and cognitive function: 25 years of insight from anatomy and neuroimaging.
      ,
      • Wang D.
      • Buckner R.L.
      • Liu H.
      Cerebellar asymmetry and its relation to cerebral asymmetry estimated by intrinsic functional connectivity.
      ) and the notable asymmetry in functional specialization in the cerebellum (
      • Petersen S.E.
      • Fox P.T.
      • Posner M.I.
      • Mintun M.
      • Raichle M.E.
      Positron emission tomographic studies of the processing of singe words.
      ). A low OT concentration (
      • Hashimoto H.
      • Fukui K.
      • Noto T.
      • Nakajima T.
      • Kato N.
      Distribution of vasopressin and oxytocin in rat brain.
      ) and the projection of afferent OT fibers (
      • Gimpl G.
      • Fahrenholz F.
      The oxytocin receptor system: Structure, function, and regulation.
      ) in the cerebellum have been reported in rats, but there is currently no evidence in humans. However, the cerebellum is increasingly being recognized to play an important role in aspects of social cognition requiring high levels of abstraction in humans (
      • Van Overwalle F.
      • Baetens K.
      • Marien P.
      • Vandekerckhove M.
      Social cognition and the cerebellum: A meta-analysis of over 350 fMRI studies.
      ). This role is consistent with the presence of anatomic and functional cerebellar abnormalities in autistic spectrum disorders in which social dysfunction is one of the core symptoms (
      • Fatemi S.H.
      • Aldinger K.A.
      • Ashwood P.
      • Bauman M.L.
      • Blaha C.D.
      • Blatt G.J.
      • et al.
      Consensus paper: Pathological role of the cerebellum in autism.
      ). Further studies are required to elucidate the exact causes of the observed IN-OT effects in rCBF in the cerebellum.
      Changes in rCBF at rest induced by IN-OT showed a predominantly left hemisphere laterality (and consistently with a contralateral cerebral-cerebellar mapping, the right cerebellum). This finding is remarkably consistent with the meta-analytic evidence that IN-OT modulated activity over a specifically left hemisphere network during processing of emotional stimuli (
      • Rocchetti M.
      • Radua J.
      • Paloyelis Y.
      • Xenaki L.A.
      • Frascarelli M.
      • Caverzasi E.
      • et al.
      Neurofunctional maps of the “maternal brain” and the effects of oxytocin: A multimodal voxel-based meta-analysis.
      ). These regions were also part of the maternal brain network responding to visual or auditory stimuli of the mother’s own (compared with unknown) children (
      • Rocchetti M.
      • Radua J.
      • Paloyelis Y.
      • Xenaki L.A.
      • Frascarelli M.
      • Caverzasi E.
      • et al.
      Neurofunctional maps of the “maternal brain” and the effects of oxytocin: A multimodal voxel-based meta-analysis.
      ). The mammalian brain, from humans to mice, shows a left hemisphere advantage in processing species-specific communication sounds (
      • Geissler D.B.
      • Ehret G.
      Auditory perception vs. recognition: Representation of complex communication sounds in the mouse auditory cortical fields.
      ). Resting-state fMRI and task-based studies have also reported hemispheric asymmetries regarding the modulatory effects of IN-OT on amygdala functional connectivity (
      • Striepens N.
      • Scheele D.
      • Kendrick K.M.
      • Becker B.
      • Schafer L.
      • Schwalba K.
      • et al.
      Oxytocin facilitates protective responses to aversive social stimuli in males.
      ) or amygdala response involving left (
      • Domes G.
      • Lischke A.
      • Berger C.
      • Grossmann A.
      • Hauenstein K.
      • Heinrichs M.
      • et al.
      Effects of intranasal oxytocin on emotional face processing in women.
      ,
      • Petrovic P.
      • Kalisch R.
      • Singer T.
      • Dolan R.J.
      Oxytocin attenuates affective evaluations of conditioned faces and amygdala activity.
      ,
      • Rilling J.K.
      • DeMarco A.C.
      • Hackett P.D.
      • Thompson R.
      • Ditzen B.
      • Patel R.
      • et al.
      Effects of intranasal oxytocin and vasopressin on cooperative behavior and associated brain activity in men.
      ,
      • Wittfoth-Schardt D.
      • Grunding J.
      • Wittfoth M.
      • Lanfermann H.
      • Heinrichs M.
      • Domes G.
      • et al.
      Oxytocin modulates neural reactivity to children’s faces as a function of social salience.
      ) or right amygdala laterality (
      • Lischke A.
      • Gamer M.
      • Berger C.
      • Grossmann A.
      • Hauenstein K.
      • Heinrichs M.
      • et al.
      Oxytocin increases amygdala reactivity to threatening scenes in females.
      ,
      • Riem M.M.
      • Bakermans-Kranenburg M.J.
      • Pieper S.
      • Tops M.
      • Boksem M.A.
      • Vermeiren R.R.
      • et al.
      Oxytocin modulates amygdala, insula, and inferior frontal gyrus responses to infant crying: A randomized controlled trial.
      ,
      • Riem M.M.
      • van I.M.H.
      • Tops M.
      • Boksem M.A.
      • Rombouts S.A.
      • Bakermans-Kranenburg M.J.
      No laughing matter: Intranasal oxytocin administration changes functional brain connectivity during exposure to infant laughter.
      ). These findings might be consistent with the noted functional asymmetry in the amygdala, with right amygdala involved in the rapid, automatic recognition of threatening stimuli and left amygdala involved in the conscious perception and regulation of the level of the emotional response (
      • Morris J.S.
      • Ohman A.
      • Dolan R.J.
      Conscious and unconscious emotional learning in the human amygdala.
      ). The reported effects of IN-OT in left amygdala in the resting state may relate to the anxiolytic effects of IN-OT in humans (
      • MacDonald K.
      • Feifel D.
      Oxytocin’s role in anxiety: A critical appraisal.
      ). A similar left hemisphere laterality has been reported regarding IN-OT modulatory effects in the striatum and frontal and temporal cortex regions (
      • Domes G.
      • Lischke A.
      • Berger C.
      • Grossmann A.
      • Hauenstein K.
      • Heinrichs M.
      • et al.
      Effects of intranasal oxytocin on emotional face processing in women.
      ,
      • Petrovic P.
      • Kalisch R.
      • Singer T.
      • Dolan R.J.
      Oxytocin attenuates affective evaluations of conditioned faces and amygdala activity.
      ,
      • Rilling J.K.
      • DeMarco A.C.
      • Hackett P.D.
      • Thompson R.
      • Ditzen B.
      • Patel R.
      • et al.
      Effects of intranasal oxytocin and vasopressin on cooperative behavior and associated brain activity in men.
      ,
      • Wittfoth-Schardt D.
      • Grunding J.
      • Wittfoth M.
      • Lanfermann H.
      • Heinrichs M.
      • Domes G.
      • et al.
      Oxytocin modulates neural reactivity to children’s faces as a function of social salience.
      ), and several nodes in the social brain network [e.g., inferior parietal cortex (
      • Radua J.
      • Phillips M.L.
      • Russell T.
      • Lawrence N.
      • Marshall N.
      • Kalidindi S.
      • et al.
      Neural response to specific components of fearful faces in healthy and schizophrenic adults.
      ) or inferior frontal gyrus (
      • Mars R.B.
      • Neubert F.X.
      • Noonan M.P.
      • Sallet J.
      • Toni I.
      • Rushworth M.F.
      On the relationship between the “default mode network” and the “social brain”.
      ,
      • Pobric G.
      • Hamilton A.F.
      Action understanding requires the left inferior frontal cortex.
      )]. The left hemisphere bias regarding IN-OT effects on brain function might reflect the known lateralization of key cognitive processes required for life in large social networks, such as communication skills and group membership categorization, to the left hemisphere (
      • Rogers L.R.
      • Vallortigara G.
      • Andrew R.J.
      Divided Brains.
      ).
      Existing histologic evidence from postmortem human brains cannot illuminate the observed asymmetries in the functional effects of IN-OT. Early studies did not report the hemispheric origin of their samples (
      • Loup F.
      • Tribollet E.
      • Dubois-Dauphin M.
      • Dreifuss J.J.
      Localization of high-affinity binding sites for oxytocin and vasopressin in the human brain. An autoradiographic study.
      ,
      • Loup F.
      • Tribollet E.
      • Dubois-Dauphin M.
      • Pizzolato G.
      • Dreifuss J.J.
      Localization of oxytocin binding sites in the human brainstem and upper spinal cord: An autoradiographic study.
      ); a later study included some bilateral but mostly left hemisphere samples (
      • Boccia M.L.
      • Petrusz P.
      • Suzuki K.
      • Marson L.
      • Pedersen C.A.
      Immunohistochemical localization of oxytocin receptors in human brain.
      ). Genetic imaging studies that investigate the effects of polymorphic variation in the OT receptor gene on brain structure (
      • Furman D.J.
      • Chen M.C.
      • Gotlib I.H.
      Variant in oxytocin receptor gene is associated with amygdala volume.
      ,
      • Inoue H.
      • Yamasue H.
      • Tochigi M.
      • Abe O.
      • Liu X.
      • Kawamura Y.
      • et al.
      Association between the oxytocin receptor gene and amygdalar volume in healthy adults.
      ,
      • Tost H.
      • Kolachana B.
      • Hakimi S.
      • Lemaitre H.
      • Verchinski B.A.
      • Mattay V.S.
      • et al.
      A common allele in the oxytocin receptor gene (OXTR) impacts prosocial temperament and human hypothalamic-limbic structure and function.
      ,
      • Tost H.
      • Kolachana B.
      • Verchinski B.A.
      • Bilek E.
      • Goldman A.L.
      • Mattay V.S.
      • et al.
      Neurogenetic effects of OXTR rs2254298 in the extended limbic system of healthy Caucasian adults.
      ,
      • Yamasue H.
      • Suga M.
      • Yahata N.
      • Inoue H.
      • Tochigi M.
      • Abe O.
      • et al.
      Reply to: Neurogenetic effects of OXTR rs2254298 in the extended limbic system of healthy Caucasian adults.
      ) and function (
      • Tost H.
      • Kolachana B.
      • Hakimi S.
      • Lemaitre H.
      • Verchinski B.A.
      • Mattay V.S.
      • et al.
      A common allele in the oxytocin receptor gene (OXTR) impacts prosocial temperament and human hypothalamic-limbic structure and function.
      ,
      • Tost H.
      • Kolachana B.
      • Verchinski B.A.
      • Bilek E.
      • Goldman A.L.
      • Mattay V.S.
      • et al.
      Neurogenetic effects of OXTR rs2254298 in the extended limbic system of healthy Caucasian adults.
      ,
      • Damiano C.R.
      • Aloi J.
      • Dunlap K.
      • Burrus C.J.
      • Mosner M.G.
      • Kozink R.V.
      • et al.
      Association between the oxytocin receptor (OXTR) gene and mesolimbic responses to rewards.
      ,
      • Jack A.
      • Connelly J.J.
      • Morris J.P.
      DNA methylation of the oxytocin receptor gene predicts neural response to ambiguous social stimuli.
      ,
      • Loth E.
      • Poline J.B.
      • Thyreau B.
      • Jia T.
      • Tao C.
      • Lourdusamy A.
      • et al.
      Oxytocin receptor genotype modulates ventral striatal activity to social cues and response to stressful life events.
      ,
      • Michalska K.J.
      • Decety J.
      • Liu C.
      • Chen Q.
      • Martz M.E.
      • Jacob S.
      • et al.
      Genetic imaging of the association of oxytocin receptor gene (OXTR) polymorphisms with positive maternal parenting.
      ,
      • Montag C.
      • Sauer C.
      • Reuter M.
      • Kirsch P.
      An interaction between oxytocin and a genetic variation of the oxytocin receptor modulates amygdala activity toward direct gaze: Evidence from a pharmacological imaging genetics study.
      ,
      • Wang J.
      • Qin W.
      • Liu B.
      • Wang D.
      • Zhang Y.
      • Jiang T.
      • et al.
      Variant in OXTR gene and functional connectivity of the hypothalamus in normal subjects.
      ) might shed further light. However, to date, there are too few studies and too great a range of tasks for a consistent pattern to emerge. Overall, these studies involve the same cortical and subcortical regions composing the oxytocinergic network identified here, including bilateral (e.g., anterior cingulate cortex) (
      • Tost H.
      • Kolachana B.
      • Verchinski B.A.
      • Bilek E.
      • Goldman A.L.
      • Mattay V.S.
      • et al.
      Neurogenetic effects of OXTR rs2254298 in the extended limbic system of healthy Caucasian adults.
      ,
      • Loth E.
      • Poline J.B.
      • Thyreau B.
      • Jia T.
      • Tao C.
      • Lourdusamy A.
      • et al.
      Oxytocin receptor genotype modulates ventral striatal activity to social cues and response to stressful life events.
      ) or predominantly left hemisphere (
      • Tost H.
      • Kolachana B.
      • Hakimi S.
      • Lemaitre H.
      • Verchinski B.A.
      • Mattay V.S.
      • et al.
      A common allele in the oxytocin receptor gene (OXTR) impacts prosocial temperament and human hypothalamic-limbic structure and function.
      ,
      • Damiano C.R.
      • Aloi J.
      • Dunlap K.
      • Burrus C.J.
      • Mosner M.G.
      • Kozink R.V.
      • et al.
      Association between the oxytocin receptor (OXTR) gene and mesolimbic responses to rewards.
      ,
      • Jack A.
      • Connelly J.J.
      • Morris J.P.
      DNA methylation of the oxytocin receptor gene predicts neural response to ambiguous social stimuli.
      ,
      • Michalska K.J.
      • Decety J.
      • Liu C.
      • Chen Q.
      • Martz M.E.
      • Jacob S.
      • et al.
      Genetic imaging of the association of oxytocin receptor gene (OXTR) polymorphisms with positive maternal parenting.
      ,
      • Wang J.
      • Qin W.
      • Liu B.
      • Wang D.
      • Zhang Y.
      • Jiang T.
      • et al.
      Variant in OXTR gene and functional connectivity of the hypothalamus in normal subjects.
      ) and right cerebellum effects (Supplement) (
      • Loth E.
      • Poline J.B.
      • Thyreau B.
      • Jia T.
      • Tao C.
      • Lourdusamy A.
      • et al.
      Oxytocin receptor genotype modulates ventral striatal activity to social cues and response to stressful life events.
      ).

      Ascertaining Temporal Dynamics of IN-OT Effects on Human Brain Physiology

      The application of GPC on rCBF maps reflecting the distributed effects of IN-OT or placebo yielded two main findings. First, classification accuracies were significant for scans after administration compared with baseline scans at all temporal intervals for the IN-OT group, but not the placebo group: predictive probabilities for the former were significantly higher than predictive probabilities for the latter. This finding suggests that IN-OT-induced changes in rCBF—and hence neuronal metabolism (
      • Cha Y.H.
      • Jog M.A.
      • Kim Y.C.
      • Chakrapani S.
      • Kraman S.M.
      • Wang D.J.
      Regional correlation between resting state FDG PET and pCASL perfusion MRI.
      ) and activity (
      • Attwell D.
      • Buchan A.M.
      • Charpak S.
      • Lauritzen M.
      • Macvicar B.A.
      • Newman E.A.
      Glial and neuronal control of brain blood flow.
      ,
      • Sokoloff L.
      Relationships among local functional activity, energy metabolism, and blood flow in the central nervous system.
      ,
      • Raichle M.E.
      • Grubb Jr, R.L.
      • Gado M.H.
      • Eichling J.O.
      • Ter-Pogossian M.M.
      Correlation between regional cerebral blood flow and oxidative metabolism. In vivo studies in man.
      ,
      • Roland P.E.
      • Eriksson L.
      • Stone-Elander S.
      • Widen L.
      Does mental activity change the oxidative metabolism of the brain?.
      ,
      • Hirano Y.
      • Stefanovic B.
      • Silva A.C.
      Spatiotemporal evolution of the functional magnetic resonance imaging response to ultrashort stimuli.
      )—were sustained over the entire observation interval.
      Physiologically, this finding is consistent with evidence from animal studies that endogenous or exogenous OT binds on hypothalamic OT-secreting neurons initiating energy-demanding processes that induce prolonged effects on physiology and behavior that last >1 hour (
      • Ludwig M.
      • Leng G.
      Dendritic peptide release and peptide-dependent behaviours.
      ,
      • Stoop R.
      Neuromodulation by oxytocin and vasopressin.
      ,
      • Sabatier N.
      • Rowe I.
      • Leng G.
      Central release of oxytocin and the ventromedial hypothalamus.
      ,
      • Sabatier N.
      • Caquineau C.
      • Dayanithi G.
      • Bull P.
      • Douglas A.J.
      • Guan X.M.
      • et al.
      Alpha-melanocyte-stimulating hormone stimulates oxytocin release from the dendrites of hypothalamic neurons while inhibiting oxytocin release from their terminals in the neurohypophysis.
      ). Human studies provide indirect support: an acute dose of IN-OT (compared with placebo) leads to an elevated concentration of OT in the CSF at 75 minutes in adult male volunteers (
      • Striepens N.
      • Kendrick K.M.
      • Hanking V.
      • Landgraf R.
      • Wullner U.
      • Maier W.
      • et al.
      Elevated cerebrospinal fluid and blood concentrations of oxytocin following its intranasal administration in humans.
      ) and for 7 hours after administration in saliva (
      • van Ijzendoorn M.H.
      • Bhandari R.
      • van der Veen R.
      • Grewen K.M.
      • Bakermans-Kranenburg M.J.
      Elevated salivary levels of oxytocin persist more than 7 h after intranasal administration.
      ,
      • Weisman O.
      • Zagoory-Sharon O.
      • Feldman R.
      Intranasal oxytocin administration is reflected in human saliva.
      ), possibly by engaging hypothalamic OT neurons in a feed-forward loop. However, the peripheral and central release of OT are dissociated (
      • Ludwig M.
      • Leng G.
      Dendritic peptide release and peptide-dependent behaviours.
      ); plasma and CSF OT levels do not correlate in humans (
      • Amico J.A.
      • Tenicela R.
      • Johnston J.
      • Robinson A.G.
      A time-dependent peak of oxytocin exists in cerebrospinal fluid but not in plasma of humans.
      ,
      • Jokinen J.
      • Chatzittofis A.
      • Hellstrom C.
      • Nordstrom P.
      • Uvnas-Moberg K.
      • Asberg M.
      Low CSF oxytocin reflects high intent in suicide attempters.
      ,
      • Kagerbauer S.M.
      • Martin J.
      • Schuster T.
      • Blobner M.
      • Kochs E.F.
      • Landgraf R.
      Plasma oxytocin and vasopressin do not predict neuropeptide concentrations in human cerebrospinal fluid.
      ,
      • Altemus M.
      • Fong J.
      • Yang R.
      • Damast S.
      • Luine V.
      • Ferguson D.
      Changes in cerebrospinal fluid neurochemistry during pregnancy.
      ), and peripheral OT cannot cross the blood-brain barrier in sufficient quantities to induce central changes (
      • Modi M.E.
      • Connor-Stroud F.
      • Landgraf R.
      • Young L.
      • Parr L.
      Aerosolized oxytocin increases cerebrospinal fluid oxytocin in rhesus macaques.
      ). Our findings suggest that changes in rCBF provide a quantifiable, reliable index to link peripheral changes in OT concentration with central effects following IN-OT.
      Our second finding was that the temporal profile of IN-OT-induced rCBF changes showed a peak response 39–51 minutes after IN-OT, followed by a gradual diminution of effects. This finding matches the slow pharmacokinetics of OT in the CSF (
      • Mens W.B.
      • Witter A.
      • van Wimersma Greidanus T.B.
      Penetration of neurohypophyseal hormones from plasma into cerebrospinal fluid (CSF): Half-times of disappearance of these neuropeptides from CSF.
      ) and is remarkably consistent with the dynamic changes in OT concentration in the extracellular fluid in the amygdala and hippocampi in rodents that peaked 30–60 minutes after the intranasal application of OT (
      • Neumann I.D.
      • Maloumby R.
      • Beiderbeck D.I.
      • Lukas M.
      • Landgraf R.
      Increased brain and plasma oxytocin after nasal and peripheral administration in rats and mice.
      ). The release of endogenous OT also shows a similar pattern in rodents when triggered with alpha-melanocyte-stimulating hormone, peaking ~20–30 minutes after stimulation (
      • Sabatier N.
      • Caquineau C.
      • Dayanithi G.
      • Bull P.
      • Douglas A.J.
      • Guan X.M.
      • et al.
      Alpha-melanocyte-stimulating hormone stimulates oxytocin release from the dendrites of hypothalamic neurons while inhibiting oxytocin release from their terminals in the neurohypophysis.
      ).

      Limitations

      Using independent groups for the IN-OT and placebo arms and concealing the identity of the nasal spray until debriefing offered protection against OT-related expectation effects (e.g., as might have arisen from differential exposure to media hype about expected outcomes of OT). In a crossover design, the absence of perceivable changes in subjective experience following IN-OT (
      • MacDonald E.
      • Dadds M.R.
      • Brennan J.L.
      • Williams K.
      • Levy F.
      • Cauchi A.J.
      A review of safety, side-effects and subjective reactions to intranasal oxytocin in human research.
      ) might have led participants to think that they had received placebo. However, our independent groups design may have inflated the between-subject variance, which precluded cross-group PR. Given the nature of this study, conducted at rest and by treatment-naïve radiographers, with minimal interaction between the main investigator and participants, a single-blind design (where the main investigator was not naïve regarding the administered compound) was deemed sufficient. Finally, we focused on male participants because some degree of sexual dimorphism in the OT system (
      • Domes G.
      • Lischke A.
      • Berger C.
      • Grossmann A.
      • Hauenstein K.
      • Heinrichs M.
      • et al.
      Effects of intranasal oxytocin on emotional face processing in women.
      ,
      • Lischke A.
      • Gamer M.
      • Berger C.
      • Grossmann A.
      • Hauenstein K.
      • Heinrichs M.
      • et al.
      Oxytocin increases amygdala reactivity to threatening scenes in females.
      ,
      • Macdonald K.S.
      Sex, receptors, and attachment: A review of individual factors influencing response to oxytocin.
      ) may be expected, and most participants in experimental studies (
      • MacDonald E.
      • Dadds M.R.
      • Brennan J.L.
      • Williams K.
      • Levy F.
      • Cauchi A.J.
      A review of safety, side-effects and subjective reactions to intranasal oxytocin in human research.
      ) and clinical trials [e.g., in autistic spectrum disorders (
      • Anagnostou E.
      • Soorya L.
      • Chaplin W.
      • Bartz J.
      • Halpern D.
      • Wasserman S.
      • et al.
      Intranasal oxytocin versus placebo in the treatment of adults with autism spectrum disorders: A randomized controlled trial.
      ,
      • Andari E.
      • Duhamel J.R.
      • Zalla T.
      • Herbrecht E.
      • Leboyer M.
      • Sirigu A.
      Promoting social behavior with oxytocin in high-functioning autism spectrum disorders.
      ,
      • Dadds M.R.
      • MacDonald E.
      • Cauchi A.
      • Williams K.
      • Levy F.
      • Brennan J.
      Nasal oxytocin for social deficits in childhood autism: A randomized controlled trial.
      ,
      • Domes G.
      • Heinrichs M.
      • Kumbier E.
      • Grossmann A.
      • Hauenstein K.
      • Herpertz S.C.
      Effects of intranasal oxytocin on the neural basis of face processing in autism spectrum disorder.
      ,
      • Gordon I.
      • Vander Wyk B.C.
      • Bennett R.H.
      • Cordeaux C.
      • Lucas M.V.
      • Eilbott J.A.
      • et al.
      Oxytocin enhances brain function in children with autism.
      ,
      • Lin I.F.
      • Kashino M.
      • Ohta H.
      • Yamada T.
      • Tani M.
      • Watanabe H.
      • et al.
      The effect of intranasal oxytocin versus placebo treatment on the autonomic responses to human sounds in autism: A single-blind, randomized, placebo-controlled, crossover design study.
      )] are male, aiming to maximize the applicability and resource efficiency for this novel study. Our findings need to be replicated in a double-blinded, crossover design including both genders.

      Conclusions

      Our findings are consistent with animal evidence and extend this evidence to humans. Our findings provide the experimenter and clinician with direct evidence to guide decision making, guide research in the pharmacokinetics and pharmacodynamics of IN-OT, and inform the development of theoretical and mechanistic accounts regarding effects of OT on typical and atypical social behavior. The power of arterial spin labeling to quantify the effects of IN-OT on brain physiology renders it a promising, noninvasive, in vivo method to investigate the impact of genetic (
      • Skuse D.H.
      • Lori A.
      • Cubells J.F.
      • Lee I.
      • Conneely K.N.
      • Puura K.
      • et al.
      Common polymorphism in the oxytocin receptor gene (OXTR) is associated with human social recognition skills.
      ,
      • Bakermans-Kranenburg M.J.
      • van Ijzendoorn M.H.
      A sociability gene? Meta-analysis of oxytocin receptor genotype effects in humans.
      ), epigenetic (
      • Jack A.
      • Connelly J.J.
      • Morris J.P.
      DNA methylation of the oxytocin receptor gene predicts neural response to ambiguous social stimuli.
      ), social-environmental (
      • Riem M.M.
      • van Ijzendoorn M.H.
      • Tops M.
      • Boksem M.A.
      • Rombouts S.A.
      • Bakermans-Kranenburg M.J.
      Oxytocin effects on complex brain networks are moderated by experiences of maternal love withdrawal.
      ), and contextual (
      • Bartz J.A.
      • Zaki J.
      • Bolger N.
      • Ochsner K.N.
      Social effects of oxytocin in humans: Context and person matter.
      ) factors on the baseline function of the OT system. Additionally, the quantification of IN-OT effects on brain physiology will allow the establishment of dose-response associations between achieved effects on neurophysiology and behavior or clinical symptoms. This information will contribute to enhancing the validity and reliability of clinical trials investigating the therapeutic potential of IN-OT and cannot be obtained using the nominal dosage of extant nasal sprays because it does not reliably reflect tissue absorption (
      • Guastella A.J.
      • Hickie I.B.
      • McGuinness M.M.
      • Otis M.
      • Woods E.A.
      • Disinger H.M.
      • et al.
      Recommendations for the standardisation of oxytocin nasal administration and guidelines for its reporting in human research.
      ).

      Acknowledgments And Disclosures

      This work was supported by Economic and Social Research Council fellowship Grant No. ES/K009400/1 (to YP), the Volkswagen Foundation “European Platform for Life Sciences, Mind Sciences and Humanities” Grant No. II/85 069 (to AF), Institute for the Study of Affective Neuroscience/Hope for Depression Research Foundation (to AF) , European Research Council Starting Investigator Award Grant No. ERC-2012-STG GA313755 (to AF), Innovative Medicines Initiative Joint Undertaking under Grant Agreement No. 115008 (NEWMEDS consortium) (to OMD), and Medical Research Council Developmental Pathway Funding Scheme Grant No. MR/J005142/1 (to MAH and SCW). The Innovative Medicines Initiative Joint Undertaking is a public-private partnership between the European Union and the European Federation of Pharmaceutical Industries and Associations. We thank our participants and Dr. D. Alsop for facilitating the pulsed-continuous arterial spin labeling pulse sequence used in this work. We also thank the National Institute for Health Research, Biomedical Research Centre for Mental Health at South London and Maudsley National Health Service Foundation Trust and Institute of Psychiatry, King’s College London for their continued infrastructure support of our neuroimaging research. The authors report no biomedical financial interests or potential conflicts of interest.

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