Effects of oxytocin administration and conditioned oxytocin on brain activity: An fMRI study.

It has been demonstrated that secretion of several hormones can be classically conditioned, however, the underlying brain responses of such conditioning have never been investigated before. In this study we aimed to investigate how oxytocin administration and classically conditioned oxytocin influence brain responses. In total, 88 females were allocated to one of three groups: oxytocin administration, conditioned oxytocin, or placebo, and underwent an experiment consisting of three acquisition and three evocation days. Participants in the conditioned group received 24 IU of oxytocin together with a conditioned stimulus (CS) during three acquisition days and placebo with the CS on three evocation days. The oxytocin administration group received 24 IU of oxytocin and the placebo group received placebo during all days. On the last evocation day, fMRI scanning was performed for all participants during three tasks previously shown to be affected by oxytocin: presentation of emotional faces, crying baby sounds and heat pain. Region of interest analysis revealed that there was significantly lower activation in the right amygdala and in two clusters in the left superior temporal gyrus in the oxytocin administration group compared to the placebo group in response to observing fearful faces. The activation in the conditioned oxytocin group was in between the other two groups for these clusters but did not significantly differ from either group. No group differences were found in the other tasks. Preliminary evidence was found for brain activation of a conditioned oxytocin response; however, despite this trend in the expected direction, the conditioned group did not significantly differ from other groups. Future research should, therefore, investigate the optimal timing of conditioned endocrine responses and study whether the findings generalize to other hormones as well.

RCT Entities:   Population Interventions Outcomes

Introduction

It has been shown that after repeated administration of medication that triggers a physiological change (unconditioned response), with an initially neutral conditioned stimulus (CS; such as, a taste or smell of the medication or the medication administration procedure), the CS alone can cause this physiological change [1]. This principle is known as pharmacological conditioning. It has been proposed that physiological responses to a CS help organisms to adapt their state in preparation for an upcoming change and in this way maintain homeostasis [2]. The principle of pharmacological conditioning has been demonstrated for various hormonal and immune parameters. For example, some evidence indicates that cortisol levels can be decreased by presenting participants with a distinctive drink previously coupled with a sumatriptan injection [3] and increased by giving a placebo injection that was previously coupled with dexamethasone [4]. Evidence of the effects of pharmacological conditioning also exists for other hormones, such as growth hormone [4] and insulin [5], and for immune parameters, such as interleukin-2 [6], natural cell killer activity [7] and histamine [8].

Despite extensive research in the field of pharmacological conditioning, no studies so far investigated neural mechanisms underlying this phenomenon. From the pain conditioning literature, it is known that conditioned analgesia decreases brain activation in pain-sensitive brain regions, such as the thalamus, insula, and dorsal anterior cingulate cortex [9]. It can be hypothesized that, similarly to the pain conditioning findings, pharmacological conditioned responses might trigger similar brain areas that are activated by the unconditioned stimulus (i.e., the medication).

In the present study, we examined for the first time the neural underpinnings of pharmacological conditioning with oxytocin. Oxytocin is a peptide hormone produced in the hypothalamus and is found to have a wide range of effects on brain activity. Its receptors are densely situated in the hypothalamus, amygdala, olfactory bulbs, and cingulate cortex [10], areas that are also associated with maternal care, social attachment and emotional processing [11]. It has been repeatedly demonstrated that exogenously administered oxytocin modifies brain responses to emotional [12, 13] and aversive visual stimuli [14], motivational tasks, involving trust [15], empathy [16], reward [17], and pain [18-20]. Particularly, oxytocin has been shown to reduce amygdala activation in response to aversive [14] and painful stimuli [20], which can be an underlying mechanism of stress reducing [21] and analgesic [22] effects of oxytocin described in previous research. Considering these positive physiological and psychological effects of oxytocin, pharmacological conditioning of oxytocin might have important clinical implications both in somatic and mental health. In this randomized placebo-controlled trial, we investigated the effects of oxytocin administration and conditioned oxytocin in comparison to a placebo-control group on brain activation in response to fMRI tasks that have previously shown to be affected by exogenous oxytocin administration: presentation of emotional faces [12], presentation of crying baby sounds [23] and thermal pain stimulation [18, 20]. We expected that the conditioned oxytocin group would demonstrate comparable brain activation patterns as the group that received exogenous oxytocin. Particularly, in response to the presentation of emotional faces, we expected that exogenous and conditioned oxytocin would reduce the activation in bilateral amygdala, and increase the activation in the insula, the occipital fusiform gyrus, and the superior temporal gyrus. We also expected that exogenous and conditioned oxytocin would decrease activation in the bilateral amygdala and increase activation in the insula and the inferior frontal gyrus pars triangularis in response to the sounds of crying babies. Finally, we expected that exogenous and conditioned oxytocin would decrease activation in the bilateral amygdala in response to pain stimulation. We also hypothesized that the changes in brain activation triggered by conditioned oxytocin, would be smaller in magnitude than the changes cause by exogenous oxytocin administration.

Materials and methods

Participants

This study is part of a study on the effects of pharmacological conditioning of oxytocin effects in which in total 99 healthy female volunteers were included [24]. Of this initial sample, 88 participants took part in the MRI part of this study (11 participants did not continue with the last part due to health and planning reasons). Participants were randomly (based on a 1:1:1 ratio with a block randomization and a block size of 8) assigned to three groups: an oxytocin administration group (29 participants), a conditioned oxytocin group (29 participants), and a placebo control group (30 participants). Participants were screened for the following exclusion criteria: intake of analgesic and anti-inflammatory medication at the moment of the experiment, psychiatric, somatic, severe neurological or neurosurgical conditions that could interfere with the participant's safety or the study protocol, left-handedness, non-removable metal parts in the body, claustrophobia, (intended) pregnancy or breast feeding, and heavy use of alcohol or drugs. Only female participants were included into the study as the effects of oxytocin on brain activation have been shown to differ between the sexes [25, 26], and although this choice limits generalizability of the findings it enhances statistical power. Moreover, only participants who used oral contraceptives were included in the trial to have a better control of menstrual cycle related hormonal changes [27]. Participants were scanned in the weeks when they used oral contraceptives, not in their stop week. Participants were asked to refrain from drinking alcohol and doing intense physical exercise 24 hours before the sessions and drinking caffeinated drinks two hours before the sessions.

The study was approved by the Medical Ethical Committee of Leiden University Medical Centre (NL52683.058.15). All participants gave written informed consent to participate in the experiment and were debriefed and financially compensated afterwards.

Sample size

The sample size was calculated with software G*Power 3. The calculation was done on the basis of a pilot experiment on conditioning of cortisol responses performed in our lab, as the design of this pilot corresponded to the design of the present study. The effect size found in the pilot experiment was d = 0.527. It was shown that 33 participants per group were necessary to obtain a power of .95 at an alpha level of a = .05. The power analysis was aimed at the question of the possibility to condition oxytocin release and not on the fMRI part of the trial.

The number of participants excluded at each step of the experiment is presented on Fig 1. One participant was not able to perform the faces task due to a technical problem with the computer. The data of one participant from the conditioned group was excluded from the analysis due to excessive head motion (frame displacement > 1 mm on 50 slices) leaving data of 86 participants that were included in the analysis of the faces task (29 participants in the oxytocin administration group, 28 participants in the oxytocin conditioned group, 29 participants in the placebo group).

Fig 1

CONSORT flow diagram.

Due to a technical problem with the audio system, 5 participants were not able to perform the crying baby sounds task. Additionally, data of 5 participants were excluded due to excessive head motion (frame displacement > 1 mm on 75–322 slices). Data of 78 participants in total were therefore included into the analysis of the crying baby sounds task (26 oxytocin administration group, 23 conditioned oxytocin group, 29 placebo group).

Due to technical problems with the thermode, 74 of 88 participants could take part in the pain task. Data of all of them were included in the analysis of the pain task (24 oxytocin administration group, 25 conditioned oxytocin, 25 placebo group).

Study design

The study had a single-blind design. Participants were randomly allocated to one of the three groups and did not know whether they received the oxytocin or the placebo spray. Researchers knew which participants were included in the oxytocin administration group (due to the absence of the CS in the evocation phase). However, researchers stayed blinded regarding the conditioned oxytocin and placebo groups. The randomization was performed by the Clinical Pharmacy of Leiden University Medical Centre using block randomization. The researchers received the randomization list after the study was completed. The trial was preregistered as a clinical trial on www.trialregister.nl (number NTR5596).

Procedures

The detailed procedures of the trial have been described elsewhere [24]. Briefly, after an initial screening, participants were randomly allocated to an oxytocin administration group, a conditioned oxytocin group, or a placebo control group. In line with previous conditioning studies [28, 29], a two-phase conditioning paradigm with an acquisition phase and an evocation phase was used. Both phases lasted for three consecutive days with a four-day break in between to allow wash-out of potential residual oxytocin effects from the previous phase. In the conditioned oxytocin group, the procedure was the following: in the acquisition phase, an association between a US (24 IU of oxytocin nasal spray) and a CS (a smell of rosewood oil) was established. Participants were administered the oxytocin spray which was immediately preceded and immediately followed by an odor of rosewood oil that was presented via a custom-made olfactometer (Wiff Online). In the evocation session, participants were administered a placebo spray paired with the same odor as in the acquisition phase. Participants in the placebo group underwent the same procedures but instead of the oxytocin spray they received a placebo spray during both phases. Participants in the oxytocin administration group received the oxytocin spray during both acquisition and evocation phases, however, they did not receive a CS during the evocation phase in order to avoid the occurrence of a conditioned response. The MRI experiment described in this study was performed on the third (last) evocation day in order to keep the conditioning context stable through the first experimental sessions.

On this third evocation day, upon arrival to the lab, participants were asked to provide a baseline saliva sample to measure their baseline oxytocin levels. Afterwards, placebo nasal spray with the odor of rosewood oil or oxytocin spray without the odor was administered, depending on group allocation. Five minutes after the spray administration, participants gave a second saliva sample and went to another lab (5-minute walk) to participate in the MRI part of the experiment.

The MRI scanning started approximately 50 minutes after the spray administration. First, an anatomical scan was performed. This was followed by several functional scans in a fixed order: the emotional faces task, the crying sounds task and finally the pain task. In total, scanning lasted for around 50 minutes.

The data were collected in the laboratory facilities of Leiden University and Leiden University Medical Centre. The data were collected between February, 2016 and August, 2017.

Oxytocin analysis

Oxytocin levels were measured in saliva. Each sample contained a minimum of 1.5 ml saliva that was collected with a passive drool method. Commercial ELISA kits with extraction (Enzo Life Sciences, Farmingdale, NY) were used for assaying salivary oxytocin. Lower level of detection for oxytocin was 0.5 pg/ml after extraction. Extraction efficiency was 99%. Intra-assay coefficient of variation was 10.2%. Inter-assay coefficient of variation was 11.8%.

Faces task

Color photographs of males and females with four different emotional facial expressions (neutral, fearful, happy, and angry) from the Radboud Faces Database [30] were used. The pictures were grouped in blocks of 5 male and 5 female pictures of each particular valence. Each picture was presented for 1300 milliseconds with an interstimulus interval of 100 milliseconds and a block duration of 13.9 seconds. A total number of 16 blocks (4 blocks of each valence) were presented in randomized order with inter-block intervals of 10 seconds (total time of the stimulation: 7.56 minutes or 454 seconds). Stimulus presentation and response registration were controlled using E-Prime 2.0 software (Psychology Software Tools, Pittsburgh, PA).

During this task, participants were asked to focus on the screen and observe blocks of photos with faces. They were subsequently asked to rate the emotional arousal of each block on a scale from 1 (not arousing at all) to 4 (very arousing). The rating was done during the between block pause of 10 seconds. Participants could provide their ratings using button boxes placed on their thighs that were within easy reach.

Crying baby sounds task

The crying baby sounds task used in the current study was similar to the one that has been extensively described in previous studies [23, 31]. The crying sounds were recorded from a 2-day old child. The control sounds were digitally created identical to the crying sounds in terms of duration, intensity, spectral content, and amplitude envelope but lacking an emotional meaning [31]. The sounds were presented in 48 blocks (24 crying sounds and 24 control sounds) of 10 seconds with 6 seconds in between in randomized order. The order of the blocks was randomized within each participant. Participants were asked to focus on the sounds during the task.

Pain task

Pain stimuli were delivered with a standardized heat pain application device (fMRI-compatible ATS thermode attached to a Pathway device, Medoc Advanced Medical Systems, Ramat Yishai, Israel). The ATS thermode was applied to the dorsal site of the left arm of the participants when they were lying in the scanner. Before performing the functional scan, but within the scanner room, the temperature that elicited a medium pain intensity of 6 on a 0 to 10 numeric rating scale (0- no pain at all; 10- the worst pain imaginable) was identified for each participant. For this purpose, participants received a sequence of ascending temperatures with a peak temperature lasting for 5 seconds and an inter-stimulus interval of 15 seconds. Participants were asked to rate each stimulus using a 0 (no pain) -10 (worst pain imaginable) Numerical Rating Scale (NRS). The temperature that was rated as eliciting pain of 6 was used subsequently during the following functional MRI.

The pain task immediately followed the individual calibration phase and consisted of alternating 7 heat pain stimuli with a peak temperature lasting for 15 seconds, and 6 baseline stimuli of neutral to slightly warm (32 degrees Celsius) temperatures lasting between 13 and 17 seconds (on average 15 seconds) each. The purpose of the variable inter-stimulus times was to avoid anticipatory pain responses. Participants were instructed to focus on the sensation they experienced and were given the opportunity to stop the task at any moment by pressing an alarm bell. No participants pressed the alarm bell during the experiment.

Image acquisition

The MRI data were acquired on a Philips 3T MR-system (Best, The Netherlands) in the Leiden University Medical Centre. First, a T1 weighted high resolution anatomical scan was acquired (repetition time (TR) = 9.8 milliseconds, echo time (TE) = 4.6 milliseconds, flip angle = 8°; voxel size 0.875 x 0.875 x 1.2; 140 slices). Functional data were acquired with echoplanar images (EPI) using a T2*-weighted gradient echo sequence (TR = 2200 milliseconds; TE = 30 milliseconds; flip angle = 80°; voxel size 2.75 × 2.75 × 2.75 millimetres +10% slice gap, 38 transverse slices). Images were scanned parallel to the anterior–posterior commissure plane.

Statistical analysis of demographic and psychological variables

Group differences in age, BMI, and baseline oxytocin salivary levels from the screening and three evocation days were examined using one-way analysis of variance (ANOVA).

To investigate whether there was significant conditioned oxytocin release, the conditioned oxytocin group was compared to the placebo group without adding the exogenous oxytocin group into the analysis (as extremely high oxytocin levels were expected in the exogenous oxytocin group which was decided prior to the study in the study registration protocol). The comparison was done with three (for each evocation day separately) repeated measures analyses of covariance (ANCOVA) with baseline oxytocin levels as a covariate. Next, the oxytocin administration group was added to the analyses and the three groups were compared on salivary oxytocin levels after the spray administration with repeated measures analyses of covariance in which baseline oxytocin levels served as a covariate.

To investigate whether exogenous or conditioned oxytocin had an effect on the arousal ratings given during the Faces task, arousal ratings of faces were compared between the groups with a factorial 4 (face valence: neutral, happy, angry or fearful) x 3 (group: oxytocin administration, placebo, conditioned oxytocin) ANOVA.

Finally, an ANOVA was used to compare the groups on the temperatures that elicited a pain of 6 (on an 11-point NRS) that were used during the Pain task.

Image preprocessing and analyses

The data were pre-processed and analysed with FSL software Version 5.0.10 (FMRIB’s Software Library, www.fmrib.ox.ac.uk/fsl [32]). Brain extraction from the anatomical scans was done using the Brain Extraction Tool as implemented in FSL [33]. Motion correction of functional scans was done using MCFLIRT [34]. Spatial smoothing was applied using a Gaussian kernel of full-width-at-half-maximum = 5 mm. High-pass temporal filtering was applied to the data (for faces task with high pass filter cut-off = 60 seconds; for crying baby sounds task filter = 50 seconds; for pain task filter = 90 seconds). Functional scans were registered to T1 weighted images, using Boundary-Based Registration, and then registered to an MNI-152 standard space image (Montreal Neurological Institute, Montreal, QC, Canada) using non-linear registration with a warp resolution of 10 millimetres.

The analysis consisted of three levels. The first level analysis was performed using general linear models. Blocks (for the faces task: angry, happy, fearful, neutral; for the crying baby sounds task: crying sounds, control sounds; for the pain task: painful stimuli, control stimuli) were used as predictors and convolved with a double-gamma hemodynamic response function and its temporal derivatives. Regression coefficients were estimated in FSL. For the faces task, six contrasts were estimated: angry >/< neutral, happy >/< neutral, fearful >/< neutral. For the crying baby sounds task, two contrasts were estimated: cry >/< control. For the pain task, two contrasts were estimated: pain >/< control. These first level contrasts were submitted to the second level analysis that was separately run per group. Within the second level analysis, the effects of the tasks on the brain activation were investigated in each group separately. Finally, to investigate the differences between the three groups, the third level analysis was performed. The three groups were compared with each other using analysis of variance on the contrasts. The Randomise tool of FSL was used to perform voxel-wise permutation-based non-parametric testing and generate statistical inference for the analysis of variance. 5000 permutations per contrast were done. The statistical tests were corrected for multiple comparisons with the threshold-free cluster enhancement (TFCE). A TFCE corrected statistical threshold of p < 0.05 was chosen.

For exploratory purposes, these analyses were first performed on the whole brain (as in Domes and colleagues [35] and Riem and colleagues [31]). Second, a region of interest analysis was run. The regions of interest were chosen based on previous literature. Based on a recent meta-analysis on the effects of oxytocin on the brain activity in response to emotion processing tasks [26], the following regions of interest were chosen for the faces task: the bilateral amygdala, the bilateral insula, the bilateral occipital fusiform gyrus and bilateral superior temporal gyrus. For the crying baby sounds task, the bilateral amygdala, bilateral insula and the bilateral inferior frontal gyrus pars triangularis were chosen as regions of interest [31, 36]. For the pain task, left and right amygdala separately were chosen as areas of interest [20]. The masks for the regions of interest were taken from the Harvard-Oxford Cortical and Subcortical Structural Atlases (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/data/atlas-descriptions.html).

Results

Baseline characteristics

There were no significant differences between the three groups on age (F (2, 87) = 0.495, p = 0.611) and body mass index (F (2, 87) = 1.083, p = 0.343); the average age across the groups was 21.5 years (SD = 2.4) and mean BMI was 22.38 (SD = 2.4).

Salivary oxytocin levels

The salivary levels of the whole sample (99 participants) during all experimental days are presented elsewhere [24]. Here we present the data of the sample of 88 participants who were included in the MRI part of the experiment. Due to clogging of the saliva samples (i.e., the saliva was thickened and could not be analyzed), 48 samples could not be analysed while 832 samples were included in the analysis. Mean salivary oxytocin levels across the groups and measurement moments and the number of analysed samples per group are presented in Table 1.There were no significant differences between the three groups on baseline salivary oxytocin levels on the screening (F (2, 80) = 1.01, p = .369), evocation day 1 (F (2, 83) = 1.47, p = 0.234), 2 (F (2, 84) = 0.37, p = 0.964) and 3 (F (2, 83) = 0.53, p = 0.588). There was a significant difference between the conditioned oxytocin and placebo groups in the levels of oxytocin after the CS administration on the evocation day1: the conditioned oxytocin group had higher salivary oxytocin levels in comparison to the placebo group after controlling for the baseline levels (F (1, 55) = 5.98, p = 0.02). No differences between conditioned oxytocin and placebo groups were found on the salivary oxytocin level on the evocation day 2 (F (1, 55) = 1.84, p = 0.18) and evocation day 3 (F (1, 55) = 1, p = 0.32). When the oxytocin administration group was added to the analysis, a significant main effect of group was found on evocation day 1 (F (2, 79) = 14.92, p < 0.001), evocation day 2 (F (2, 79) = 15.29, p < 0.001) and evocation day 3 (F (2, 80) = 11.68, p < 0.001). Post hoc Bonferroni comparison demonstrated that the oxytocin administration group had significantly higher salivary oxytocin levels than the placebo and the conditioned oxytocin group after controlling for the baseline levels during all evocation days (all p’s < .001).

Table 1

Mean salivary oxytocin levels (pg/ml) and standard deviations (SD) across the groups and measurement moments.

Test day	Measurement	Placebo group	Oxytocin administration group	Conditioned oxytocin group
Screening	Baseline	12.57 (SD = 12.62, n = 30)	9.66 (SD = 6.66, n = 25)	15.32 (SD = 20.08, n = 26)
Evocation day 1	Baseline	16.94 (SD = 19.64, n = 30)	12.21 (SD = 7.05, n = 26)	11.68 (SD = 8.97, n = 28)
	+ 5 minutes	14.85 (SD = 7.65, n = 30)	1912.79 (SD = 2429.66, n = 25)	28.34 (SD = 44.63, n = 28)
	+ 20 minutes	13.77 (SD = 8.47, n = 30)	1020.81 (SD = 1860.49, n = 25)	20.17 (SD = 28.76, n = 28)
	+ 50 minutes	10.18 (SD = 5.1, n = 30)	848.15 (SD = 1569.4, n = 26)	21.17 (SD = 25.99, n = 28)
Evocation day 2	Baseline	13.23 (SD = 8, n = 30)	13.01 (SD = 9.32, n = 27)	13.24 (SD = 7.12, n = 28)
	+ 5 minutes	13.97 (SD = 6.6, n = 30)	1727.86 (SD = 2272.05, n = 25)	30.89 (SD = 70, n = 28)
	+ 20 minutes	13.14 (SD = 5.62, n = 30)	1102.87 (SD = 1650.52, n = 25)	17.36 (SD = 15.37, n = 28)
	+ 50 minutes	11.08 (SD = 5.66, n = 30)	826.88 (SD = 1666.11, n = 26)	10.14 (SD = 7.55, n = 28)
Evocation day 3	Baseline	16.85 (SD = 21.56, n = 30)	14.84 (SD = 13.6, n = 26)	12.14 (SD = 12.91, n = 28)
	+ 5 minutes	12.19 (SD = 7.49, n = 30)	1719.83 (SD = 2639.64, n = 24)	20.24 (SD = 43.93, n = 27)

Faces task

First, to examine the effects of the emotional faces’ stimuli on brain activation, we looked at the second level analysis, with a specific focus on the placebo group to see the effects of the task on the brain activity regardless of the effects of oxytocin. The results of the second level analysis are presented in Table 2 (for all three groups separately) and in the Supporting Information (S1–S4 Figs). Full brain analysis in the placebo group (first column in Table 2) revealed a significant activation in two clusters in the right and left occipital fusiform gyrus, one cluster in the right superior temporal gyrus and one cluster in the right inferior temporal gyrus for the contrast fearful > neutral faces. The ROI analysis additionally showed clusters in the right amygdala, the right insula, the bilateral occipital fusiform gyrus and the bilateral superior temporal gyrus that were significantly active on the contrast fearful > neutral faces. Additionally, heightened activation in the left occipital fusiform gyrus was found on the contrast happy > neutral faces in the ROI analysis.

Table 2

Effects of face valence across the groups (second level analysis).

	Placebo group (n = 29)					Oxytocin administration group (n = 29)					Conditioned oxytocin group (n = 28)
	Cluster size	T max	X (mm)	Y (mm)	Z (mm)	Cluster size	T max	X (mm)	Y (mm)	Z (mm)	Cluster size	T max	X (mm)	Y (mm)	Z (mm)
1. Neutral < angry
ROI left amygdala						2	6.17	-34	-8	-20
ROI left OFG											23	4.21	-22	-88	-10
1. Neutral > angry
ROI right insula						20	4.31	34	-14	-2
2. Neutral < happy
WB left occipital pole											5	4.71	0	-92	-6
WB left occipital pole superior devision											50	5.61	-16	-98	4
WB right occipital pole											34	5.27	12	-102	6
											16	6.41	16	-102	-2
ROI left OFG extending to occipital pole	4	4.36	-18	-96	-4						746	4.91	-6	-92	-10
3.Neutral < fearful
WB bilateral OFG						4573	8.18	22	-90	-6
WB left OFG extending to lateral occipital cortex	591	6.01	-28	-86	-12						16	4.79	-36	-84	-10
											200	5.38	-22	-80	-10
WB right OFG extending to lateral occipital cortex	232	5.15	28	-88	-10						376	6.25	22	-88	-10
WB right STG posterior division	123	4.83	46	-36	4
WB right inferior temporal gyrus, temporooccipital part	24	4.29	46	-52	-8
ROI right amygdala	16	3.39	28	6	-22
ROI right insula	22	3.5	36	26	0
ROI bilateral OFG	3679	6.01	-28	-86	-12	4535	8.18	22	-90	-6	869	5.38	-22	-88	-10
											1088	6.25	22	-88	-10
ROI left OFG	60	4.76	-46	-12	-12
ROI right OFG	253	4.83	46	-36	4

WB- results obtained with the whole brain analysis; ROI- results obtained with the region of interest analysis; OFG- occipital fusiform gyrus; STG- superior temporal gyrus. Reported activations are corrected for multiple comparisons with the threshold-free cluster enhancement. Coordinates are reported using the Montreal Neurologic Institute space.

In the third level brain analysis we compared the three groups with each other. The whole brain analysis of variance with all three groups (oxytocin administration, conditioned oxytocin and placebo) demonstrated no significant differences in any of the three contrasts. The ROI analysis revealed that there was significantly higher activation in the right amygdala in the placebo group in comparison to the oxytocin administration group on the contrast fearful > neutral. To explore this result in detail, we plotted z-values and standard deviations of the three groups for this significant cluster (Fig 2). The z-values of the conditioned oxytocin group were in between the values of the oxytocin administration and placebo groups but did not significantly differ from either of these groups. Additionally, ROI analysis yielded a significant difference between placebo and oxytocin administration group in the left superior temporal gyrus: the activation in the oxytocin administration group was significantly lower than in the placebo group on the contrast fearful > neutral (Fig 3). The Z-values of the conditioned oxytocin group were, again, in between the values of the oxytocin administration and placebo groups but did not significantly differ from either of these groups. No significant activation was found in other regions of interest.

Fig 2

Cluster in right amygdala, contrast neutral < fearful.

Cluster (28, 8, -24; t max = 3.81, cluster size = 4) with the significantly lower activation in the oxytocin group in comparison to the placebo group on the contrast neutral < fearful and Z statistics with standard deviations from this cluster.

Fig 3

Clusters in left superior temporal gyrus, contrast neutral < fearful.

Clusters in the left superior temporal gyrus (cluster 1: -50, -32, 0; t max = 4.23, cluster size = 41; cluster 2: -46, -10, -12; t max = 4.84; cluster size = 14) with the significantly lower activation in the oxytocin group in comparison to the placebo group on the contrast neutral < fearful and mean Z statistics with standard deviations from these clusters.

No differences were found between the groups in the arousal ratings given during the task (F (2, 77) = 0.15, p = .858). There was a significant difference in how arousing participants found faces of different modality (F (3, 77) = 116.85, p < .001). Bonferroni corrections demonstrated there were significant differences between all couples of modalities (all p’s < .05) and that happy faces were found to be the most arousing (M = 3.00), followed by fearful faces (M = 2.75), and angry faces (M = 2.50). Neutral faces (M = 1.23) were rated as the least arousing.

Crying baby sounds task

The results of the second level analysis of the Sounds task are presented in Table 3 and in the Supporting Information (S5–S7 Figs). First, we again explored the effects of the sounds on the brain activation in the placebo group alone (first column of Table 3). The whole brain analysis showed higher activation in one large cluster in the right superior temporal gyrus with extension to the planum polare and one cluster in the left planum polare on the cry > control sounds contrasts. Subsequent ROI analysis showed that the cry > control sounds contrast caused significant activation in the right and left amygdala, the right and left insula and the left inferior frontal gyrus pars triangularis. The third level analysis with the comparison between the three groups revealed neither significant differences in the whole brain level nor in the ROI analyses.

Table 3

Effects of the sound valence across the groups (second level analysis).

	Placebo group (n = 29)					Oxytocin group (n = 26)					Conditioned group (n = 23)
	Cluster size	T max	X (mm)	Y (mm)	Z (mm)	Cluster size	T max	X (mm)	Y (mm)	Z (mm)	Cluster size	T max	X (mm)	Y (mm)	Z (mm)
Cry > control
WB right STG with extension to right planum polare	2980	9.75	58	-10	-2	1185	7.3	64	-14	2	33	5.6	64	-24	4
WB left planum polare	2489	10.1	-54	-4	2	1034	6.99	-50	-10	4
ROI left amygdala	15	3.92	-22	-6	-14
ROI right amygdala	45	5.09	32	8	-20
ROI left insula	310	7.64	-50	-2	-2	94	6.26	-46	-8	0
ROI right insula	300	7.56	50	4	-4	69	6.15	50	-4	-4
ROI left IFG, pars triangularis	56	3.75	44	32	4

WB- results obtained with the whole brain analysis; ROI- results obtained with the region of interest analysis; STG- superior temporal gyrus; IFG = inferior frontal gyrus. Reported activations are corrected for multiple comparisons with the threshold-free cluster enhancement. Coordinates are reported using the Montreal Neurologic Institute space.

Pain task

The results of the second level analysis of the Pain task are presented in Table 4 and in the Supporting Information (S1–S4 Figs). The effects of pain stimulation on brain activity were again first examined in the placebo group alone (first column of the Table 4). The whole brain analysis revealed significant activation in 12 clusters across the brain on the contrast pain > control and in 1 cluster on the contrast control > pain (for the details see Table 4). Significantly increased activation in both the left and right amygdala were found on the ROI analysis on the contrast pain > control. The third level analysis, comparing the three groups, revealed no significant effect of oxytocin administration or conditioning with oxytocin on brain activity neither in the whole brain analysis nor in the ROI analysis with left and right amygdala.

Table 4

Effects of the pain stimulation across the groups (second level analysis).

	Placebo group (n = 25)					Oxytocin group (n = 23)					Conditioned group (n = 25)
	Cluster size	T max	X (mm)	Y (mm)	Z (mm)	Cluster size	T max	X (mm)	Y (mm)	Z (mm)	Cluster size	T max	X (mm)	Y (mm)	Z (mm)
pain>control
WB right postcentral gyrus with extension to precentral gyrus	10776	6.82	48	-26	64	455	6.36	48	-20	58	21	4.81	60	-4	28
WB left temporal occipital fusiform cortex	1786	5.23	-28	-38	-24
WB left temporal occipital fusiform cortex with extension to left occipital fusiform gyrus						27763	8.18	-26	-46	-10
WB right parahippocampal gyrus with extension to temporal fusiform cortex	623	6.77	26	-24	-24						32709	7.79	22	-22	-18
WB bilateral occipital pole	248	5.19	0	-88	40
WB right lateral occipital cortex	217	4.89	44	-68	26
WB bilateral precuneous cortex	162	4.15	10	-60	14
WB left thalamus	118	4.8	-14	-32	10
WB left middle temporal gyrus	87	4.35	-68	-8	-10						25	4.01	-52	-12	-18
WB brain stem	81	4.68	-2	-26	-22
WB angular gyrus	33	3.45	48	-54	30
WB right cingulate gyrus	21	3.57	8	-50	6
WB bilateral cuneal cortex	15	3.26	-2	-84	36
WB frontal pole with extension to right paracingulate gyrus											1212	5.54	0	66	4
WB left superior frontal gyrus											278	5.12	-24	22	42
ROI left amygdala	78	4.53	-20	-10	-24						18	5.03	-20	-16	-18
	26	4.37	-26	-2	-32
ROI right amygdala	38	4.55	14	-4	-24						173	4.17	24	2	-26
pain < control
WB right precentral gyrus	837	6.52	46	10	26
WB left precentral gyrus						23	5.55	-58	6	4
WB right frontal operculum cortex to insula and central opercular cortex						1484	11	48	18	0
WB right central opercular cortex											121	5.79	52	0	8
WB right frontal pole						68	5.53	40	38	8
WB right insular cortex											422	6.95	34	12	10

WB- results obtained with the whole brain analysis; ROI- results obtained with the region of interest analysis. Reported activations are corrected for multiple comparisons with the threshold-free cluster enhancement. Coordinates are reported using the Montreal Neurologic Institute space.

Finally, there were no significant differences between the three groups on the temperature that was used during the Pain task (oxytocin administration group: M = 46.2, SD = 1.1; conditioned oxytocin group: M = 46.4, SD = 1.2; placebo group: M = 45.3, SD = 1.6; F (2, 73) = 0.537, p = 0.587).

Discussion

This is the first study that investigated the effects of pharmacological conditioning with oxytocin on brain activity. We hypothesized that conditioned oxytocin responses would demonstrate patterns of brain activation similar to exogenous oxytocin administration and that both these groups would differ from the placebo group. Differences in the brain activation between the oxytocin administration and placebo groups were demonstrated in the right amygdala and in two clusters in superior temporal gyrus for the emotional faces task only, while brain activation of the conditioned oxytocin group was in between that of the oxytocin administration and the placebo group but did not significantly differ from either group.

The amygdala has been shown to play a role in negative emotion processing [37] and its activation has been found in response to threat [38]. Previous research has demonstrated that presentation of faces with fearful expression increases amygdala activation and 24 IU oxytocin has been repeatedly shown to dampen this effect [12, 13, 39]. We replicated these previous findings and furthermore showed that there is a careful indication that conditioning with oxytocin might slightly affect this activity pattern as well but to a lesser extent than exogenous oxytocin. However, since no significant differences between the conditioned group and other two groups were found, this finding should be interpreted cautiously.

Moreover, the same fearful > neutral contrast yielded a significant difference between the oxytocin administration and placebo group in two clusters of the superior temporal gyrus (STG). The STG plays an important role in the processing of emotional stimuli and social cognition [40] and particularly processing of fearful faces [12, 41]. With this finding we thus replicated previous results showing increased STG activity in response to the presentation of the fearful faces. However, the direction of this oxytocin effect does not correspond to most previous studies. Several previous studies showed enhanced STG activity in response to emotional and social stimuli after oxytocin administration [26, 42]. However, in our study we found that participants in the oxytocin group had lower activation in the STG on the contrast fear > neutral in comparison to the placebo group. Some studies, also found dampening effects of oxytocin on STG activation. For example, a decrease in STG activity after oxytocin administration was found in response to social rejection [43]. Also, Hech and colleagues [44] demonstrated that oxytocin reduced brain activation to social stimuli and, particularly, that individuals with higher levels of social processing exhibited oxytocin induced decrease in STG in response to social stimuli. The findings on the directionality of STG brain activity in response to oxytocin are thus mixed in the current literature. In our study, we found an increase in STG in response to fearful faces in the placebo condition and this increase was dampened by oxytocin in the oxytocin condition, corresponding to our findings in the amygdala. Again, STG activity in the conditioned oxytocin group was in between the oxytocin and placebo groups but did not significantly differ from both groups. Possibly, similar to the results of the study on social rejection [43], oxytocin inhibited the processing of negative emotions of fearful faces in our study.

The significant differences were found between oxytocin administration and placebo groups in these two areas, and at the same time the activation in the conditioned group was in between these two groups and did not significantly differ from them. This finding might be indicative of a smaller response of the conditioned group in comparison to the effect of oxytocin administration, however this should be interpreted with caution. Possibly, there was not enough power in the between-subject design of this study to find this small effect.

The sounds of a crying baby activated the auditory cortex in all groups and the amygdala and the inferior frontal gyrus, pars triangularis in the placebo group, as was expected [29]. Even though significant activation by the cry > control contrast was found in the amygdala and inferior frontal gyrus pars triangularis in the placebo group and not in the oxytocin administration and conditioned oxytocin groups on the second level analyses, the between-group comparison in the third level analysis did not reach significance. A previous study [31] found that oxytocin reduced activation in the amygdala and increased activation in the insula and the inferior frontal gyrus pars triangularis on the contrast cry > control. We could not replicate these results. Speculatively, this could be due to design differences, as Riem and colleagues [31] included twins in their sample, and performed the task 45 minutes after the oxytocin administration while our task was done approximately 60 minutes after the spray (as it followed the faces task).

The pain task activated large clusters across the brain, including primary and secondary somatosensory cortex, thalamus, cingulate gyrus and amygdala, the areas that have been repeatedly shown to be activated by acute pain [45, 46]. Importantly, several studies showed that oxytocin affects brain responses to experimentally induced pain and particularly dampens amygdala activation [18, 20, 46]. Indeed, the increased activation on the contrast pain > control in the left and right amygdala was found only in the placebo group and not in the conditioned and oxytocin administration groups in the second level analyses, but the between-group comparison was not significant. Possibly, the effects of both exogenous and conditioned oxytocin were not strong enough to be seen in the between-group comparison. The evidence about the effects of oxytocin on brain activation in response to pain, is, not conclusive. Singer and colleagues [18] found that oxytocin decreases amygdala activation in response to heat pain stimuli, however, they proposed that the effects were driven by selfish participants: effects of oxytocin on the amygdala activation were found only in selfish, but not prosocial participants. Another study by Zunhammer and colleagues [19] did not find effects of oxytocin on brain activity in response to heat pain. Speculatively, oxytocin might influence emotional or social aspects of pain perception that have not been captured by our study as, for example, it has been shown that oxytocin enhances the pain-relieving effects of social support [47] and affects neural activity while seeing pain in others [48].

The results of both the crying baby sounds and pain task showed that the second level analyses are partially in line with the previous literature as heightened amygdala activation in response to the crying sounds and pain stimulation was found in the placebo group but not in the oxytocin administration group. However, the effects were not strong enough to be seen in the between-group comparisons. One possible explanation for this lack of significance can be the timing of the experiment. We conducted the MRI scanning on the third evocation day to avoid interference with the conditioning procedure, because it has been previously shown that presenting a distinctive additional stimulus during the conditioning might inhibit the conditioned response [49] and the whole MRI environment can be perceived to be stressful and distracting. On the third evocation day, the conditioned response in saliva had already been extinguished even though it was found on the first evocation day [24]. Since salivary oxytocin levels might not be the only indicator of the conditioned response, we still hypothesized that we could observe the conditioned response in the brain. Speculatively, if the fMRI experiment was done on the first evocation day, a stronger response in the brain might have been found. Future studies are needed to confirm this hypothesis. Moreover, the fMRI scan started approximately 50 minutes after the oxytocin and placebo administration. This time frame was chosen because the neuronal effects of exogenous oxytocin administration have been demonstrated to be the strongest around this time [50]. However, the conditioned response does not necessarily correspond to the timing of the effects of exogenous oxytocin administration. The only study on endocrine conditioning that investigated the conditioned response temporally was a study on conditioned insulin release [5] which found that the conditioned insulin release appeared around 40 minutes after the first placebo administration. However, insulin and oxytocin are distinct systems and the results from insulin conditioning cannot necessarily be directly generalized to the timing of oxytocin conditioning. As the highest conditioned oxytocin levels in saliva were found immediately after the conditioned stimulus administration on the first evocation day [24], the strongest response in the brain might possibly also immediately follow the conditioned stimulus administration and may already have decreased 50 minutes later. The fact that the only difference between the groups was found on the first task and not on the subsequent tasks supports this speculation to some extent. For future studies, it is advised that the effects of oxytocin conditioning are studied on the first evocation day, and possibly immediately following the placebo administration with repeated measurements across time to find the peak of the conditioned response.

Another possible explanation of the non-significant effects of conditioned oxytocin release on brain activity, is the difference in the magnitude of the effects between exogenous oxytocin administration and the conditioned response. Our data shows that even during evocation day 1, when the largest conditioned response was found in saliva, the oxytocin levels increased twice from the baseline, compared to a 100-time increase in the oxytocin administration group. It is unknown whether salivary oxytocin increase corresponds to the change in the brain activity in a linear way, but it can be expected that the neural effects of conditioned oxytocin release might be much smaller than the effects of oxytocin administration. However, even small natural variations of the endogenous oxytocin levels have been shown to affect brain activity, for example, in resting state [51], during massage [52], and in response to aversive stimuli [53]. Therefore, it could be expected that endogenous oxytocin release triggered by conditioning, would be strong enough to affect brain activity.

Changing hormonal levels with a behavioral manipulation can have important clinical implications especially for disorders related to dysfunction of the endocrine system. For example, it has been demonstrated that immunosuppressive treatment for renal transplant patients can be enhanced by using classically conditioned immunosuppression [28]. Nevertheless, classically conditioned endocrine responses have not been investigated in clinical practice. The possibility to induce classically conditioned insulin release as demonstrated by Stockhorst, de Fries [5], might for example be applied for improving therapies for patients with diabetes type-2 who suffer from dysfunctional insulin release. Classical conditioning of oxytocin responses could be tested in populations with mental disorders related to emotional deficits, such as autism, schizophrenia and borderline personality disorder as oxytocin has been shown to have promising effects for treatments of these disorders [54, 55, 56]. Knowing what brain areas are involved in endocrine conditioning might be helpful for making the effects of the endocrine conditioning stronger and manipulating these effects more optimally.

To conclude, our study was the first study investigating the effects of classical conditioning with oxytocin on brain activity. We have found preliminary indications that the conditioned oxytocin response might affect the activity in the left STG and amygdala in a direction similar to exogenous oxytocin, however future studies are needed to confirm this hypothesis as no significant group difference between conditioned and other two groups was found in the current study. Moreover, these effects were not generalizable across the tasks. Future research should explore different time frames of the conditioned oxytocin brain responses and extend this paradigm to the conditioning of other hormones as comparisons to oxytocin conditioning. Unraveling the neural mechanisms of endocrine conditioning might help us to implement this potentially beneficial mechanism in clinical practice.

Second level whole brain analysis in the placebo group in the faces task on the contrast neutral < fearful.

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Second level whole brain analysis in the oxytocin group in the faces task on the contrast neutral < fearful.

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Second level whole brain analysis in the conditioned group in the faces task on the contrast neutral < fearful.

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Second level whole brain analysis in the conditioned group in the faces task on the contrast and neutral < happy.

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Second level whole brain analysis in the placebo group in the crying sounds task on the contrast control < cry.

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Second level whole brain analysis in the oxytocin group in the crying sounds task on the contrast control < cry.

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Second level whole brain analysis in the conditioned group in the crying sounds task on the contrast control < cry.

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Second level whole brain analysis in the placebo group in the pain task on the contrast control < pain.

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Second level whole brain analysis in the placebo group in the pain task on the contrast control > pain.

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Second level whole brain analysis in the oxytocin group in the pain task on the contrast control < pain.

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Second level whole brain analysis in the oxytocin group in the pain task on the contrast and control > pain.

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Second level whole brain analysis in the conditioned group in the pain task on the contrast control < pain.

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Second level whole brain analysis in the conditioned group in the pain task on the contrast control > pain.

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Click here for additional data file.

10 Dec 2019

PONE-D-19-27410

Effects of oxytocin administration and conditioned oxytocin on brain activity: an fMRI study.

PLOS ONE

Dear Ms Skvortsova,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #1: Yes

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Reviewer #3: Yes

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Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: This is an interesting and carefully-designed study. Given the intense interest in the therapeutic applications of oxytocin for various psychiatric conditions, the possibility of a conditioned oxytocin response is intriguing and potentially of great clinical value. The authors have followed many best practices for oxytocin research, including consideration of female participants’ menstrual cycle/hormonal contraceptive use and saliva sampling to confirm no baseline oxytocin differences between groups. Another strength is the authors’ choice to use 3 distinct fMRI tasks previously shown to be influenced by exogenous oxytocin, even though they only found a significant difference in 1 task. The analysis of the fMRI data is appropriate and the authors adequately address the discrepant findings in the direction of STG activation under the oxytocin condition in the Faces task. I do hope the authors continue to pursue this line of research to obtain further evidence that a conditioned oxytocin response is indeed possible.

There are several areas of the paper that the authors should further address:

1. Abstract. The findings are stated too strongly. Specifically, the following sentence should be rephrased: “The findings carefully suggest that a conditioned response in brain activity was observed, however the conditioned group did not significantly differ from the other groups”. If there is no significant difference between the conditioned group and placebo group, you cannot say that a conditioned response was observed. Especially as this non-significant difference was only found in 1 of 3 tasks. The authors should consider describing this as preliminary evidence.

2. Salivary analysis / Table 1

The author states that 48 saliva samples could not be analyzed because they were "clogged" (p 11). It is not clear what this means. It is also relevant what groups these samples belong to, and for this reason Table 1 should include sample sizes. In Table 1, I assume the values in parentheses ( ) are the standard deviations? This is not stated. While I understand that the details of the salivary hormone analysis are presented elsewhere, given on-going concerns about salivary oxytocin measurements,the authors should briefly described the method of analysis (ie what kit was used, whether there was an extraction step).

3 Discussion (page 30)

Page 30, line 409: “On the third evocation day, the conditioned response in saliva had already been extinguished”.

The data in Table 1 does not appear to support this (Day 3, +5 min, Placebo Group 12.19 pg/ml vs Conditioned Oxytocin Group 61.23 pg/ml); In fact, the oxytocin level at +5 min in the Conditioned Oxytocin Group is 2-fold greater on Evocation Day 3 vs Evocation Day 1.

The data in Table 1 also appears to conflict with this statement “As the highest conditioned oxytocin levels in saliva were found immediately after the conditioned stimulus administration on the first evocation day” (p 31, line 422). As this statement is the basis for the further suggestion that at a greater difference in brain activation may have been observed if scanning was done on Day 1, this should be clarified.

4. Given that any conditioned oxytocin release would be much smaller than the supraphysiological dose (24 IU) of intranasal oxytocin (which is supported by your salivary hormone data), how much of an effect would you expect more naturalistic oxytocin release to have on fMRI brain activation? This might be worth discussing, as it could help explain the non-significant differences between the conditioned oxytocin group and placebo group on fMRI tasks.

5. While the overall quality of the writing is acceptable, there are small errors throughout. The authors should carefully proofread their article once more. Errors I noted include:

p 7, line 153 “data was”

p 9,line 204-205 “the oxytocin salivary …”

p 10, line 246, “regions of interests”

p 13, line 292 “conditioned oxytocin [group]”

p 21, line 324 “the table 4|”

Reviewer #2: This is an interesting, elegant and well-conducted study and a well-written manuscript. It involves an ambitious and unique oxytocin conditioning experiment, performed in three relatively large samples of adult females, implying three well-established fMRI experiments.

I definitely recommend the study for publication. I do have a number of relatively minor suggestions for further improving the report.

- Page 4, end of Introduction: It would be helpful for the reader if you could already give a brief preview on the expected patterns of major activation for each of the three fMRI paradigms, and the particular hypotheses concerning the impact of exogenous/conditioned oxytocin.

- Page 4, Participants: What is the rationale for exclusively including female participants?

- Page 4, line 92-94: “Only participants who used oral contraceptives during active use weeks …’ � unclear phrasing; please rephrase

- Page 9, Preprocessing, line 221: Unclear why the first level analysis was performed in native space? + how these data have than be brought to MNI space for the second- and third-level analysis?

- Page 10, line 238-245, concerning the choice for ROIs: For the FACES task, in addition to more general emotion processing ROIs the authors also include “modality specific” ROIs, such as fusiform gyrus and superior temporal gyrus. However, for the CRYING task and for the PAIN task, the authors do not include these modality specific ROIs, i.e. auditory processing and somatosensory processing, respectively. I think it would have been more logical if they would also have performed an ROI analysis in primary and secondary auditory regions for the CRYING tasks, and in primary and secondary somatosensory regions for the PAIN task. This more focused approach might possibly reveal additional group differences for these both tasks, as already suggested by the reported cluster characteristics drawn from the whole-brain analyses in Tables 3 and 4.

- Results FACES task: Could the authors also report the behavioural data for these tasks? Were there any group differences in perceived arousal of the emotional faces?

- Page 14, Table 2 (+ also Table 3-4): Please, verify the textual presentation in this Table and in the Legend, as it is not optimal (sometimes capital letters, sometimes not). WF � WB?

- P. 30 line 410: typo “be”

- Generally, for the three fMRI paradigms: It would be informative to have whole brain plots/figures of the most informative contrasts (i.e. fear>neutral; cry>sound; pain>control); at least for the Placebo group, but preferably for the three groups separately.

Reviewer #3: This study examines the neural underpinnings of pharmacological conditioning with oxytocin. The authors tested intranasal and conditioned oxytocin effects on brain activity in response to three fMRI tasks that has been previously been shown to be affected by intranasal oxytocin administration. This study is very interesting and novel in that there is a lack of fMRI research on conditioned oxytocin effects. However, I do have some questions/comments and concerns with the way the results are presented.

Introduction:

The rationale for using a pain stimulation task to examine neural underpinnings of oxytocin conditioning is unclear, because there is no strong evidence that oxytocin affects pain sensitivity. In the introduction, the authors refer to a thermal pain stimulation task (line 79) that has been shown to be affected by intranasal oxytocin. However, in the discussion they mention that fMRI findings on oxytocin and pain perception/sensitivity are inconsistent. It is unclear why the authors expect to find effects of conditioned oxytocin during this task. Please provide a clear rationale.

Methods/results:

-The description of the power analysis is unclear. Please provide more details.

-Description of statistical analyses (line 203-207) is unclear. Please reframe.

-Were there any group differences in baseline OT before the acquisition phase?

-Grammatical error line 261 – 262: “was found” should be “were found”.

-Were there any effects of conditioned oxytocin or intranasal oxytocin on the stimulus ratings (faces and cry sounds)?

Discussion:

The authors should be more careful with interpreting insignificant results throughout the manuscript. E.g. line 444-447 � the authors mention that “effects remained small”, but effects were non-significant.

The authors found that intranasal oxytocin reduced STG activity, whereas previous studies point to enhanced STG activity. The authors should explain this discrepancy more clearly and discuss how their findings relate to previous research.

In the discussion, the authors could elaborate more on differential effects of exogenous oxytocin and manipulated endogenous oxytocin on brain activity. The increase in oxytocin induced by the conditioning paradigm is very small compared to the enormous increase induced by intranasal administration (Table 1). The authors do not find significant differences between the placebo group and conditioned oxytocin group. I wonder whether this may be due to the small increase in oxytocin, which may be insufficient to result in detectable brain activity changes.

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Reviewer #1: No

Reviewer #2: Yes: Bart Boets

Reviewer #3: No

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23 Jan 2020

January 23, 2020

Leiden, the Netherlands

Dear Prof. Dr. Bos,

We would like to thank you for the opportunity to submit a revised version of our manuscript, “Effects of oxytocin administration and conditioned oxytocin on brain activity: an fMRI study” for publication in PlosOne. We have revised the original manuscript to address the comments and concerns raised by the reviewers. The point-to-point responses to each comment are given below and changes in the manuscript are marked with Track Changes. We hope that with this revision we satisfactory addressed all issues.

We look forward to hearing from you soon.

Sincerely, on behalf of all co-authors,

Aleksandrina Skvortsova, PhD

Corresponding author

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Response: We thank the editorial office for this remark. We have added the information regarding the ethical approval to the Methods section on page 5. Now it reads as follows:

The study was approved by the Medical Ethical Committee of Leiden University Medical Centre (NL52683.058.15). All participants gave written informed consent to participate in the experiment and were debriefed and financially compensated afterwards.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

Response: The data will be made available on DataVerse.nl

Reviewer #3: Yes

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is an interesting and carefully-designed study. Given the intense interest in the therapeutic applications of oxytocin for various psychiatric conditions, the possibility of a conditioned oxytocin response is intriguing and potentially of great clinical value. The authors have followed many best practices for oxytocin research, including consideration of female participants’ menstrual cycle/hormonal contraceptive use and saliva sampling to confirm no baseline oxytocin differences between groups. Another strength is the authors’ choice to use 3 distinct fMRI tasks previously shown to be influenced by exogenous oxytocin, even though they only found a significant difference in 1 task. The analysis of the fMRI data is appropriate and the authors adequately address the discrepant findings in the direction of STG activation under the oxytocin condition in the Faces task. I do hope the authors continue to pursue this line of research to obtain further evidence that a conditioned oxytocin response is indeed possible.

Response: We thank the reviewer for the supportive words and the interest in our study.

There are several areas of the paper that the authors should further address:

1. Abstract. The findings are stated too strongly. Specifically, the following sentence should be rephrased: “The findings carefully suggest that a conditioned response in brain activity was observed, however the conditioned group did not significantly differ from the other groups”. If there is no significant difference between the conditioned group and placebo group, you cannot say that a conditioned response was observed. Especially as this non-significant difference was only found in 1 of 3 tasks. The authors should consider describing this as preliminary evidence.

Response: We thank the reviewer for this important comment. Indeed, we do not want to overstate our results in any way. We have adjusted the abstract and now it reads as follows:

Preliminary evidence was found for brain activation of a conditioned oxytocin response; however, despite this trend in the expected direction, conditioned group did not significantly differ from other groups. Future research should, therefore, investigate the optimal timing of conditioned endocrine responses and study whether the findings generalize to other hormones as well.

2. Salivary analysis / Table 1

The author states that 48 saliva samples could not be analyzed because they were "clogged" (p 11). It is not clear what this means. It is also relevant what groups these samples belong to, and for this reason Table 1 should include sample sizes. In Table 1, I assume the values in parentheses ( ) are the standard deviations? This is not stated. While I understand that the details of the salivary hormone analysis are presented elsewhere, given on-going concerns about salivary oxytocin measurements,the authors should briefly described the method of analysis (ie what kit was used, whether there was an extraction step).

Response: We thank the reviewer for these remarks. Indeed, 48 samples (5% of the total number) could not be analyzed due to clogging of the samples. It means that the saliva was thickened and could not be analyzed; this explanation is now added to the manuscript on page 12:

Due to clogging of the saliva samples (i.e., the saliva was thickened and could not be analyzed), 48 samples could not be analysed while 832 samples were included in the analysis.

We have added the number of the analyzed samples per group and per measurement moment in Table 1. We have also specified that the number in parentheses in Table 1 is indeed the standard deviation. Moreover, we have added a paragraph describing the method of oxytocin analysis to page 7. It reads as follows:

Oxytocin analysis

Oxytocin levels were measured in saliva. Each sample contained a minimum of 1.5 ml saliva that was collected with a passive drool method. Commercial ELISA kits with extraction (Enzo Life Sciences, Farmingdale, NY) were used for assaying salivary oxytocin. Lower level of detection for oxytocin was 0.5 pg/ml after extraction. Extraction efficiency was 99%. Intra-assay coefficient of variation was 10.2%. Inter-assay coefficient of variation was 11.8%.

3 Discussion (page 30)

Page 30, line 409: “On the third evocation day, the conditioned response in saliva had already been extinguished”.

The data in Table 1 does not appear to support this (Day 3, +5 min, Placebo Group 12.19 pg/ml vs Conditioned Oxytocin Group 61.23 pg/ml); In fact, the oxytocin level at +5 min in the Conditioned Oxytocin Group is 2-fold greater on Evocation Day 3 vs Evocation Day 1.

The data in Table 1 also appears to conflict with this statement “As the highest conditioned oxytocin levels in saliva were found immediately after the conditioned stimulus administration on the first evocation day” (p 31, line 422). As this statement is the basis for the further suggestion that at a greater difference in brain activation may have been observed if scanning was done on Day 1, this should be clarified.

Response: We thank the reviewer for noticing this inconsistency. There was indeed a mistake in Table 1. The oxytocin level in the conditioned group on the day 3, +5 minutes was 20.24 pg/ml and not 61.23 pg/ml. We have made the correction in Table 1 and double checked all other values. This number is supported by our statistical analysis that demonstrated that there were no differences between the conditioned and the placebo groups on this measurement point.

4. Given that any conditioned oxytocin release would be much smaller than the supraphysiological dose (24 IU) of intranasal oxytocin (which is supported by your salivary hormone data), how much of an effect would you expect more naturalistic oxytocin release to have on fMRI brain activation? This might be worth discussing, as it could help explain the non-significant differences between the conditioned oxytocin group and placebo group on fMRI tasks.

Response: We thank the review for this very interesting remark. Indeed, the conditioned response found in saliva was considerably lower than the increase in salivary oxytocin levels after the exogenous oxytocin administration. However, we do not know to what extent the increase in salivary oxytocin levels reflects the conditioned response in the brain activity. Moreover, several studies have found that natural variations in endogenous oxytocin levels predict brain activity. We have added the discussion of these issues to the discussion on page 33. Now it reads as follows:

Another possible explanation of the non-significant effects of conditioned oxytocin release on brain activity, is the difference in the magnitude of the effects between exogenous oxytocin administration and the conditioned response. Our data shows that even during evocation day 1, when the largest conditioned response was found in saliva, the oxytocin levels increased twice from the baseline, compared to a 100-time increase in the oxytocin administration group. It is unknown whether salivary oxytocin increase corresponds to the change in the brain activity in a linear way, but it can be expected that the neural effects of conditioned oxytocin release might be much smaller than the effects of oxytocin administration. However, even small natural variations of the endogenous oxytocin levels have been shown to affect brain activity, for example, in resting state [50], during massage [51], and in response to aversive stimuli [52]. Therefore, it could be expected that endogenous oxytocin release triggered by conditioning, would be strong enough to affect brain activity.

5. While the overall quality of the writing is acceptable, there are small errors throughout. The authors should carefully proofread their article once more. Errors I noted include:

p 7, line 153 “data was”

p 9,line 204-205 “the oxytocin salivary …”

p 10, line 246, “regions of interests”

p 13, line 292 “conditioned oxytocin [group]”

p 21, line 324 “the table 4|”

Response: We thank the reviewer for mentioning these errors. We have adjusted them and proofread the whole manuscript.

Reviewer #2: This is an interesting, elegant and well-conducted study and a well-written manuscript. It involves an ambitious and unique oxytocin conditioning experiment, performed in three relatively large samples of adult females, implying three well-established fMRI experiments.

I definitely recommend the study for publication. I do have a number of relatively minor suggestions for further improving the report.

Response: We thank the reviewer for the supporting words about our manuscript.

- Page 4, end of Introduction: It would be helpful for the reader if you could already give a brief preview on the expected patterns of major activation for each of the three fMRI paradigms, and the particular hypotheses concerning the impact of exogenous/conditioned oxytocin.

Response: We thank the reviewer for this advice. We have added hypotheses regarding the expected pattern of brain activation for all three tasks to the page 4:

We expected that the conditioned oxytocin group would demonstrate comparable brain activation patterns as the group that received exogenous oxytocin. Particularly, in response to the presentation of emotional faces, we expected that exogenous and conditioned oxytocin would reduce the activation in the bilateral amygdala, and increase the activation in the insula, the occipital fusiform gyrus, and the superior temporal gyrus. We also expected that exogenous and conditioned oxytocin would decrease activation in the bilateral amygdala and increase activation in the insula and the inferior frontal gyrus pars triangularis in response to the sounds of crying babies. Finally, we expected that exogenous and conditioned oxytocin would decrease activation in the bilateral amygdala in response to pain stimulation. We also hypothesized that the changes in brain activation triggered by conditioned oxytocin, would be smaller in magnitude than the changes cause by exogenous oxytocin administration.

- Page 4, Participants: What is the rationale for exclusively including female participants?

Response: It has been repeatedly shown that sex is an important moderator of the effects of oxytocin on brain activity (Rilling et al., Psychoneuroendocrinology, 2014; Grace et al., Psychoneuroendocrinology, 2018). Therefore, we decided to limit our sample to only female participants to avoid a possible confounding effect of sex differences. Moreover, this choice allowed us to enhance statistical power as we did not have to take into account a possible interaction term with sex. We have added this explanation to the manuscript on page 5:

Only female participants were included into the study as the effects of oxytocin on brain activation have been shown to differ between the sexes (25, 26), and although this choice limits generalizability of the findings it enhances statistical power.

- Page 4, line 92-94: “Only participants who used oral contraceptives during active use weeks …’ � unclear phrasing; please rephrase

Response: We have adjusted the sentence. Now it reads as follows (page 5):

Only participants who used oral contraceptives were included in the trial to have a better control of menstrual cycle related hormonal changes [27]. Participants were scanned in the weeks when they used oral contraceptives, not in their stop week.

- Page 9, Preprocessing, line 221: Unclear why the first level analysis was performed in native space? + how these data have than be brought to MNI space for the second- and third-level analysis?

Response: We thank the reviewer for this important question. The first level analysis was indeed not performed in the native space. Before the first level analysis, all functional scans were registered to T1 weighted images, using Boundary-Based Registration, and then registered to an MNI-152 standard space image; this procedure is described on page 10.

- Page 10, line 238-245, concerning the choice for ROIs: For the FACES task, in addition to more general emotion processing ROIs the authors also include “modality specific” ROIs, such as fusiform gyrus and superior temporal gyrus. However, for the CRYING task and for the PAIN task, the authors do not include these modality specific ROIs, i.e. auditory processing and somatosensory processing, respectively. I think it would have been more logical if they would also have performed an ROI analysis in primary and secondary auditory regions for the CRYING tasks, and in primary and secondary somatosensory regions for the PAIN task. This more focused approach might possibly reveal additional group differences for these both tasks, as already suggested by the reported cluster characteristics drawn from the whole-brain analyses in Tables 3 and 4.

Response: We thank the reviewer for this important remark. Choosing the ROIs for our analyses, we decided to choose a confirmative approach and focus on the areas that specifically have been shown in previous research to be affected by oxytocin administration during these specific tasks. For the task with presentation of emotional faces, we included the ROIs that have been shown to be affected by oxytocin in previous studies using this task, and these areas were amygdala, insula, fusiform gyrus and superior temporal gyrus (Domes et al., Psychoneuroendocrinology, 2010; Domes at al., Biological psychiatry, 2007; meta-analysis: Grace et al., Psychoneuroendocrinology, 2018). Therefore, by choosing fusiform gyrus and superior temporal gyrus we were not aiming to select “modality specific” ROIs but rather the ROIs that were already found in the previous research. We used the same principle in selecting ROIs for the crying sounds and pain tasks. The previous research on these two tasks did not find effects of oxytocin on the auditory cortex and somatosensory cortex respectively, and therefore, we did not include these regions in our ROI analysis. This approach we took by selecting ROIs based on previous findings is described on page 11.

- Results FACES task: Could the authors also report the behavioural data for these tasks? Were there any group differences in perceived arousal of the emotional faces?

Response: We thank the reviewer for this important question. There were no differences found between the groups in the arousal ratings. We have added the description of the analysis and results of this task to the methods on page 10 and results on page 15. Now it reads as follows:

Page10:

To investigate whether exogenous or conditioned oxytocin had an effect on the arousal ratings given during the Faces task, arousal ratings of faces were compared between the groups with a factorial 4 (face valence: neutral, happy, angry or fearful) x 3 (group: oxytocin administration, placebo, conditioned oxytocin) ANOVA.

Page 15:

No differences were found between the groups in the arousal ratings given during the task (F (2, 77) = 0.15, p = .858). There was a significant difference in how arousing participants found faces of different modality (F (3, 77) = 116.85, p < .001). Bonferroni corrections demonstrated there were significant differences between all couples of modalities (all p’s < .05) and that happy faces were found to be the most arousing (M = 3.00), followed by fearful faces (M = 2.75), and angry faces (M = 2.50). Neutral faces (M = 1.23) were rated as the least arousing.

- Page 14, Table 2 (+ also Table 3-4): Please, verify the textual presentation in this Table and in the Legend, as it is not optimal (sometimes capital letters, sometimes not). WF � WB?

Response: We thank the reviewer for these remarks. We have adjusted the Tables.

- P. 30 line 410: typo “be”

Response: We thank the reviewer for noticing this. We have corrected the typo.

- Generally, for the three fMRI paradigms: It would be informative to have whole brain plots/figures of the most informative contrasts (i.e. fear>neutral; cry>sound; pain>control); at least for the Placebo group, but preferably for the three groups separately.

Response: We thank the reviewer for this advice. We have added figures with the results of the whole brain second level analysis to the Supporting Information.

Reviewer #3: This study examines the neural underpinnings of pharmacological conditioning with oxytocin. The authors tested intranasal and conditioned oxytocin effects on brain activity in response to three fMRI tasks that has been previously been shown to be affected by intranasal oxytocin administration. This study is very interesting and novel in that there is a lack of fMRI research on conditioned oxytocin effects. However, I do have some questions/comments and concerns with the way the results are presented.

Response: We thank the reviewer for expressing interest in our study.

Introduction:

The rationale for using a pain stimulation task to examine neural underpinnings of oxytocin conditioning is unclear, because there is no strong evidence that oxytocin affects pain sensitivity. In the introduction, the authors refer to a thermal pain stimulation task (line 79) that has been shown to be affected by intranasal oxytocin. However, in the discussion they mention that fMRI findings on oxytocin and pain perception/sensitivity are inconsistent. It is unclear why the authors expect to find effects of conditioned oxytocin during this task. Please provide a clear rationale.

Response: We thank the reviewer for raising this important question. Indeed, the effects of oxytocin on pain perception remain unclear: some studies found that oxytocin decreases pain sensitivity, while others failed to confirm these results (see the systematic review: Rash et al., 2014, The Clinical journal of pain). However, we have chosen to include the heat pain task because a body of evidence points at the modulatory effects of oxytocin on the brain activation in response to pain: Zunhammer et al., 2015, Psychosomatic Medicine; Singer et al 2008, Emotion; Kreuder et al., 2019, Human Brain Mapping, for seeing pain in others: Bos et al., 2005, Neuroimage. One study that did not find modulatory effects of oxytocin on brain activity in response to pain, is the study by Zunhammer and colleagues, 2016, Scientific reports. On the basis, of the existing literature, we concluded that pain stimulation, might be a relevant task for investigating the effects of exogenous and conditioned oxytocin; however, our hypothesis was not confirmed.

We have made small modifications in the paragraph where we discuss the results of the pain task to make the rationale clearer (page 31):

The pain task activated large clusters across the brain, including primary and secondary somatosensory cortex, thalamus, cingulate gyrus and amygdala, the areas that have been repeatedly shown to be activated by acute pain [ 44, 45]. Importantly, several studies showed that oxytocin affects brain responses to experimentally induced pain and particularly dampens amygdala activation [18, 20, 46]. Indeed, the increased activation on the contrast pain > control in the left and right amygdala was found only in the placebo group and not in the conditioned and oxytocin administration groups in the second level analyses, but the between-group comparison was not significant. Possibly, the effects of both exogenous and conditioned oxytocin were not strong enough to be seen in the between-group comparison. The evidence about the effects of oxytocin on brain activation in response to pain, is however not conclusive. Singer and colleagues [18] found that oxytocin decreases amygdala activation in response to heat pain stimuli, however, they proposed that the effects were driven by selfish participants: effects of oxytocin on the amygdala activation were found only in selfish, but not prosocial participants. Another study by Zunhammer and colleagues [19] did not find effects of oxytocin on brain activity in response to heat pain. Speculatively, oxytocin might influence emotional aspects of pain perception that have not been captured by our study as, for example, it has been shown that oxytocin enhances the pain-relieving effects of social support [46] and affects neural activity while seeing pain in others [47].

Methods/results:

-The description of the power analysis is unclear. Please provide more details.

Response: We have adjusted the description of the power analysis on page 5. Now it reads as follows:

The sample size was calculated with software G*Power 3. The calculation was done on the basis of a pilot experiment on conditioning of cortisol responses performed in our lab, as the design of this pilot corresponded to the design of the present study. The effect size found in the pilot experiment was d = 0.527. It was shown that 33 participants per group were necessary to obtain a power of .95 at an alpha level of a = .05. The power analysis was aimed at the question of the possibility to condition oxytocin release and not on the fMRI part of the trial.

-Description of statistical analyses (line 203-207) is unclear. Please reframe.

Response: We have adjusted the paragraph about the statistical analysis to make it clearer (page 10):

To investigate whether there was significant conditioned oxytocin release, the conditioned oxytocin group was compared to the placebo group without adding the exogenous oxytocin group into the analysis (as extremely high oxytocin levels were expected in the exogenous oxytocin group which was d prior to the study in the study registration protocol). The comparison was done with three (for each evocation day separately) repeated measures analyses of covariance (ANCOVA) with baseline oxytocin levels as a covariate. Next, the oxytocin administration group was added to the analyses and the three groups were compared on salivary oxytocin levels after the spray administration with repeated measures analyses of covariance in which baseline oxytocin levels served as a covariate.

To investigate whether exogenous or conditioned oxytocin had an effect on the arousal ratings given during the Faces task, arousal ratings of faces were compared between the groups with factorial 4 (face valence: neutral, happy, angry or fearful) x 3 (group: oxytocin administration, placebo, conditioned oxytocin) ANOVA.

Finally, an ANOVA was used to compare the groups on the temperatures that elicited a pain of 6 (on an 11-point NRS) that were used during the Pain task.

-Were there any group differences in baseline OT before the acquisition phase?

Response: We thank the reviewer for this question. There was no difference between the groups in their salivary oxytocin levels that were measured during the screening before the first acquisition day. We have added these results to the manuscript on page 12 and to Table 1:

There were no significant differences between the three groups on baseline salivary oxytocin levels on the screening (F (2, 80) = 1.01, p = .369)...

-Grammatical error line 261 – 262: “was found” should be “were found”.

Response: we have corrected the typo.

-Were there any effects of conditioned oxytocin or intranasal oxytocin on the stimulus ratings (faces and cry sounds)?

Response: No differences were found between the groups in the arousal ratings. We have added the description of the analysis and results of this task to the methods on page 10 and results on page 15. Now it reads as follows:

To investigate whether exogenous or conditioned oxytocin had an effect on the arousal ratings given during the Faces task, arousal ratings of faces were compared between the groups with a factorial 4 (face valence: neutral, happy, angry or fearful) x 3 (group: oxytocin administration, placebo, conditioned oxytocin) ANOVA.

No differences were found between the groups in the arousal ratings given during the task (F (2, 77) = 0.15, p = .858). There was a significant difference in how arousing participants found faces of different modality (F (3, 77) = 116.85, p < .001). Bonferroni corrections demonstrated there were significant differences between all couples of modalities (all p’s < .05) and that happy faces were found to be the most arousing (M = 3.00), followed by fearful faces (M = 2.75), and angry faces (M = 2.5). Neutral faces (M = 1.23) were rated as the least arousing.

Discussion:

The authors should be more careful with interpreting insignificant results throughout the manuscript. E.g. line 444-447 � the authors mention that “effects remained small”, but effects were non-significant.

Response: We thank the reviewer for this remark. We certainly do not want to overstate the results of our study and therefore, we adjusted the sentence mentioned by the reviewer and also made some changes in the Abstract to emphasize that no group differences were found. Page 34:

Moreover, these effects were not generalizable across the tasks.

Abstract:

The activation in the conditioned oxytocin group was in between the other two groups for these clusters but did not significantly differ from either group. No group differences were found in the other tasks. Preliminary evidence was found for brain activation of a conditioned oxytocin response; however, despite this trend in the expected direction, the conditioned group did not significantly differ from other groups. Future research should, therefore, investigate the optimal timing of conditioned endocrine responses and study whether the findings generalize to other hormones as well.

The authors found that intranasal oxytocin reduced STG activity, whereas previous studies point to enhanced STG activity. The authors should explain this discrepancy more clearly and discuss how their findings relate to previous research.

Response: We thank the reviewer for the opportunity to elaborate on this issue further. Indeed, we found that oxytocin reduced STG activity in response to fearful faces, which contradicts the generally found tendency of oxytocin to enhance the activation in STG (see meta-analyses: Grace et al., 2018, Psychoneuroendocrinology; Wang et al., 2017, Social Cognitive and Affective Neuroscience). At the same time, there is also some literature showing that oxytocin might decrease the activity in STG. Hech et al. (2017, Neuroimage) found that oxytocin reduces brain activation to social stimuli and particularly, that individuals with higher levels of social processing exhibited oxytocin induced decrease in STG in response to social stimuli. Also, a decrease in the activity of STG was found in response to social rejection (Gozzi et al., 2017, Neuropsychopharmacology). Our results add to the existing conflicting evidence regarding the effects of oxytocin on STG.

We have extended the paragraph where we discuss these findings on page 30:

Moreover, the same fearful > neutral contrast yielded a significant difference between the oxytocin administration and placebo group in two clusters of the superior temporal gyrus (STG). The STG plays an important role in the processing of emotional stimuli and social cognition [40] and particularly processing of fearful faces [12, 41]. With this finding we thus replicated previous results showing increased STG activity in response to the presentation of the fearful faces. However, the direction of this oxytocin effect does not correspond to most previous studies. Several previous studies showed enhanced STG activity in response to emotional and social stimuli after oxytocin administration [26, 41]. However, in our study we found that participants in the oxytocin group had lower activation in the STG on the contrast fear > neutral in comparison to the placebo group. Some other studies also found dampening effects of oxytocin on STG activation. For example, a decrease in STG activity after oxytocin administration was found in response to social rejection [42]. Also, Hech and colleagues [43] demonstrated that oxytocin reduced brain activation to social stimuli and, particularly, that individuals with higher levels of social processing exhibited oxytocin induced decrease in STG in response to social stimuli. The findings on the directionality of STG brain activity in response to oxytocin are thus mixed in the current literature. In our study, we found an increase in STG in response to fearful faces in the placebo condition and this increase was dampened by oxytocin in the oxytocin condition, corresponding to our findings in the amygdala. Again, STG activity in the conditioned oxytocin group was in between the oxytocin and placebo groups but did not significantly differ from both groups. Possibly, similar to the results of the study on social rejection [42], oxytocin inhibited the processing of negative emotions of fearful faces in our study.

In the discussion, the authors could elaborate more on differential effects of exogenous oxytocin and manipulated endogenous oxytocin on brain activity. The increase in oxytocin induced by the conditioning paradigm is very small compared to the enormous increase induced by intranasal administration (Table 1). The authors do not find significant differences between the placebo group and conditioned oxytocin group. I wonder whether this may be due to the small increase in oxytocin, which may be insufficient to result in detectable brain activity changes.

Response: We thank the reviewer for this feedback. We have added a discussion of this issue to page 33 of the discussion:

Another possible explanation of the non-significant effects of conditioned oxytocin release on brain activity, is the difference in the magnitude of the effects between exogenous oxytocin administration and the conditioned response. Our data shows that even during evocation day 1, when the largest conditioned response was found in saliva, the oxytocin levels increased twice from the baseline, compared to a 100-time increase in the oxytocin administration group. It is unknown whether salivary oxytocin increase directly corresponds to the change in the brain activity, but it can be expected that the neural effects of conditioned oxytocin release might be much smaller than the effects of oxytocin administration. However, even small natural variations of the endogenous oxytocin levels have been shown to affect brain activity, for example, in resting state [50], during massage [51], and in response to aversive stimuli [52]. Therefore, it can be expected that endogenous oxytocin release triggered by conditioning, could be strong enough to affect brain activity.

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12 Feb 2020

Effects of oxytocin administration and conditioned oxytocin on brain activity: an fMRI study.

PONE-D-19-27410R1

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PONE-D-19-27410R1

Effects of oxytocin administration and conditioned oxytocin on brain activity: an fMRI study.

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