We analyzed non-published data acquired previously by Delplanque et al. (2009). In this study, participants were presented with a set of varied "sample" and "target" odors and given no information about them. Sample odors were presented first as an encoding condition, whereas target odors were presented second, as a retrieval condition. Only target odors were previously analyzed to be included in Delplanque et al. (2009). Here, we analyzed responses to the sample odors. Emotional responses to these odors were compared with those obtained in an independent sample of participants presented with a set of fragrances. Given the strong inter-individual variability of olfactory preferences (Herz and Von Clef, 2001; de Araujo et al., 2005; Keller et al., 2007), we conducted our analyses on the basis of individual judgments as opposed to averaging the subjective ratings for a given odor.

# Participants

Two different groups of nonsmoking participants (Group 1 and Group 2), all University of Geneva students, were recruited through ads posted in a university building. Group 1 consisted of 18 participants (9 females, right handed; mean age = 27.1 ± 6.2 years) and was provided with pleasant and unpleasant odors (Delplanque et al., 2009). Group 2 consisted of 21 participants (all females; mean age = 22.7 ± 3.3 years) and was provided with fragrances. Participants were individually tested and paid 50 Swiss Francs (approximately \$50) for their participation. On testing days, participants were asked not to wear any perfume. They all self-reported a normal sense of smell and were free from respiratory infections when they participated. None of the participants reported any mental illnesses that could have affected their emotional responses to stimuli. Written consent was obtained from all participants before starting the experiment in accordance with the Declaration of Helsinki, and the study was approved by the ethical committees of the Geneva University Hospital and of the Psychology Department of the University of Geneva. In Group 1, two participants were excluded because of acquisition artifacts in facial muscle activity (both the corrugator and the zygomaticus muscles), leaving 19 participants for analysis. In Group 2, participants were excluded because of acquisition artifacts in activities of the corrugator (one participant) and zygomaticus muscles (two participants), leaving 16 and 17 participants for analysis on these two variables, respectively.

# Stimuli

All olfactory stimuli ("*Odors*" and "*Fragrances*") were injected into the tampon of cylindric felt-tip pens (14 cm long, inner diameter 1.3 cm). The use of these highly practical devices (provided by Burghart, Germany) avoids any contamination of the environment.

#### Odors

Thirty-two *a priori* pleasant and unpleasant odorants (**Table 1**) were selected on the basis of a previous study conducted on 66 participants, who evaluated 51 odorants according to subjective intensity, pleasantness, and familiarity (see Delplanque et al., 2008, 2009). The aim of this large selection was to obtain an array of odorants with a wide pleasantness spectrum. For practical reasons, we labeled this first choice of odorants as "*Odors*".

#### Fragrances

Nine additional fine perfumes (**Table 2**) were selected on the basis of a preliminary study performed on 60 undergraduate

∗*Single odorant molecule. CAS molecule numbers are provided where available.*

TABLE 2 | Fragrances.

students (60 females; mean age = 20.27 ± 3.1 years). The primary interest of that study was to assess the influence of contextual information on fragrance evaluation. We chose fragrances that were well-known in the French and Swiss markets. In addition, the fragrances were well characterized to ensure good perceptual variability (see **Table 2**). For practical reasons, we labeled this second choice of odorants as "*Fragrances*".

#### Experimental Procedures

Participants were told that they would be provided with olfactory stimuli to evaluate. During one session, they smelled the 32 odorcontaining (Group 1, *Odors*) or the nine fragrance-containing (Group 2, *Fragrances*) pens in random order in successive trials. For each trial, an experimenter seated near the participant in a well-ventilated room then placed an odor pen about 1 cm below the participant's nostrils for 2 s. Before testing, participants were instructed via computer to smell the odorants according to a particular procedure to minimize variability in intra- and interparticipant breathing patterns (Jung et al., 2006; Delplanque et al., 2009). The participants first had to breathe out deeply through the mouth, wait for the request to inhale (a word presented on a screen in front of the participant), breathe in evenly with the felttip pen containing the odorant under the two nostrils, and then rest and relax for 15 s.

The presentation of the olfactory stimulus to the participant was followed by the completion of subjective ratings assessing intensity, hedonicity, and familiarity. The interval between two stimuli was 15 s to avoid sensory adaptation.

## Subjective Ratings

Participants rated the hedonicity, intensity, and familiarity of the olfactory stimuli that they were presented with on continuous 10 cm scales from *very unpleasant* (left of the scale = 0 cm) to *neutral* (middle of the scale, 5 cm) to *very pleasant* (right of the scale, 10 cm); from *not perceived* (or low intensity, left) to *medium* (middle) to *strong* (or high intensity, right); and from *not familiar at all* (left) to *very familiar* (right), respectively, (see Delplanque et al., 2009 for details).

#### Apparatus and Physiological Recordings

Physiological signals were assessed with the TEL 100 Remote Monitoring System (Group 1) and the MP150 (Group 2) system of Biopac (Santa Barbara, CA, USA) with separate settings for the electrocardiogram, electrodermal activity, and respiratory activities. Signals were transferred from the experimental room to the MP100 Acquisition Unit (16 bit A/D conversion) in an adjacent room and stored on computer hard disk (sampling rate 500 Hz). Respiratory activity was assessed by placing two respiration belts on the participant that measured abdominal and thoracic expansion and contraction. Electrodermal activity was recorded (high-pass filter: 0.025 Hz) by the constant-voltage method (0.5 V). Beckman Ag–AgCl electrodes (8 mm diameter active area) filled with a skin conductance paste (Biopac) were attached to the palmar side of the middle phalanges of the second and third fingers of the participants' non-dominant hand. Heart rate was assessed by fixing Biopac pregelled disposable electrodes under the participants' left and right wrists. A third electrode was placed on the left ankle. The signal was amplified by 1,000 and low-pass filtered (30 Hz). Electrocardiographic R waves were detected offline, and intervals between heartbeats were converted into heart rate, expressed in beats per minute (BPM). Surface electromyography (EMG) was collected, digitized, and stored (bandwidth 0.1 to 417 Hz, sample rate: 2,048 Hz) with a BIOSEMI Active-Two amplifier system (BioSemi Biomedical Instrumentation, Amsterdam, the Netherlands). Six active electrodes were placed over the right frontalis, corrugator, and zygomaticus regions of the face, corresponding to three distinct bipolar montages of interest (Fridlung and Cacioppo, 1986). Two additional electrodes placed above the inion (the common mode sense active electrode and the driven right leg passive electrode) were used as recording references and ground electrodes1 . Conventional bipolar montages were then calculated from electrode pairs for each muscle by subtracting the activity of one electrode placed over the muscle from the activity of the other nearby electrode in Brain Vision Analyzer software (Brain Products, Gilching, Germany). Signals were then filtered with a 20 to 400 Hz band-pass digital filter, rectified, and low-passed filtered below 40 Hz.

#### Physiological Data Analyses Respiration Parameters

The voltage amplitude of the inhalation phase during the olfactory stimulus presentation was reported and constitutes the main respiratory control.

#### Electrodermal Activity

Specific skin conductance responses (SCRs) to odors were measured in microSiemens and analyzed offline. They were scored as changes in conductance starting in the -s to 4-s interval after the beginning of inhalation (Dawson et al., 1990). SCRs were square root transformed to normalize the data (Edelberg, 1972).

#### Facial Muscle Activity

Electromyography amplitude during the 1 s before olfactory stimulus presentation served as the baseline. To allow us to examine the temporal profiles of facial EMG for 5 s after inhalation of different olfactory stimuli, we expressed mean

1http://www*.*biosemi*.*com/faq/cms&drl*.*htm

EMG amplitudes during subsequent 1 s time intervals as a percentage of the mean amplitude of the baseline. Percentage scores were introduced to standardize the widely differing absolute EMG amplitudes of individual participants and thus enable comparison between individuals and groups (e.g., de Wied et al., 2006).

#### Heart Rate

The biphasic heart response consists of cardiac acceleration peaking at about 3 s followed by a decrease in heart rate, with a minimum reached at about 6 s after the onset of inspiration (see Delplanque et al., 2009). We analyzed the maximum negative variation in the 5 to 8 s window following stimulus presentation (heart rate deceleration) to investigate whether this phase was sensitive to stimulus pleasantness. The heart rate time course during the 10 s before olfactory stimulus presentation served as the baseline. We averaged the heart rate values within successive 200 ms periods, leading to 15 heart rate scores during the 3 s interval. We then expressed these 15 heart scores as a percentage of the BPM of the baseline. Percentage scores were introduced to standardize the differing absolute BPM variations of individual participants and thus enable comparison between individuals and groups.

# Statistical Analyses

In order to obtain our intra-subject measures, two types of odors and fragrances were distinguished on the basis of each participant's own ratings: *pleasant* (two highest hedonicity scores) and *unpleasant* (two lowest hedonicity scores). We also performed correlations between the mean pleasantness rating of each odorant stimulus corresponding to a given hedonic order (1: least liked odorant to 32: most liked odorant) across individuals and the strength of its corresponding physiological response (heart rate or electrodermal response).

We computed a mixed model analysis of variance (ANOVA), with pleasantness (2: *pleasant, unpleasant*) as the within-subject repeated factor and group (2: *Odor, Fragrance*) as the between-subject factor to analyze subjective ratings, heart rate, electrodermal response, and respiratory parameters.

In the case of facial muscle activity, a Time factor (five: *0–1,1– 2,2–3,3–4,4–5 s*) was added to account for the temporal evolution of the signal, decomposed in five 1 s time intervals. We tested the significance of paired comparisons between experimental conditions, using Tukey *post hoc* comparisons (PHCs). All tests were performed by using STATISTICA 122 .

# RESULTS

# Subjective Ratings

The analysis performed on hedonicity, familiarity, and intensity ratings revealed a main effect of pleasantness on these three indicators [*F*(1,35) = 839.03, *p <* 0.001, η2 s = 0.96; *F*(1,35) = 77.98 *p <* 0.001, η2 = 0.69; and *F*(1,35) = 7.28, *p* = 0.011, η2 = 0.17, respectively]. Pleasant stimuli (*odors* and *fragrances*) were systematically evaluated as being more pleasant (average: 8.44) than unpleasant stimuli (1.81), confirming that a clear hedonic distinction was made (**Figure 1A**) based on each participant's own evaluation. Pleasant stimuli were also perceived as being more familiar (7.43) than unpleasant stimuli (4.22; **Figure 1B**). The group × pleasantness interaction was significant for both hedonic and familiarity ratings [*F*(1,35) = 55.92, *p <* 0.001, η2 = 0.61; *F*(1,35) = 12.12, *p* = 0.001, η2 = 0.26], revealing a more pronounced hedonic distinction according to pleasantness for *Odors* (PHC *p <* 0.001 for hedonicity and familiarity) compared with *Fragrances* (PHCs *p <* 0.003 for hedonicity and familiarity), since unpleasant *Odors* were rated lower than unpleasant *Fragrances* (**Figure 1A**; PHC, *p <* 0.001). This interaction was not significant for intensity ratings [*F*(1,35) = 0.14, *p* = 0.709, n.s., η2 = 0.004; **Figure 1C**],

2http://www*.*statsoft*.*com

indicating that unpleasant olfactory stimuli were more intense (7.47) than pleasant stimuli (6.92), regardless of the pleasantness spectrum (*Odors* or *Fragrances*).

## Peripheral Physiology

Group × pleasantness interactions were also observed for both peripheral physiological measures, i.e., electrodermal activity and heart rate [*F*(1,35) = 5.75, *p* = 0.022, η2 = 0.14; *F*(1,35) = 7.33, *p* = 0.010, η2 = 0.17, respectively]. Unpleasant *Odors* elicited stronger SCRs than did pleasant *Odors* (PHC, *p* = 0.033) and unpleasant *Fragrances* (PHC, *p <* 0.001; **Figure 2A**).

Unpleasant *Odors* also specifically induced a weaker heart deceleration than did pleasant *Odors* (PHC, *p* = 0.007), unlike *Fragrances* in which this effect was not significant (**Figure 2C**).

Since both interactions were significant, we performed separate regression analyses between mean hedonicity ratings and SCRs or heart rate, for *Odors* and *Fragrances*, respectively. A significant U-shaped quadratic correlation was found for *Odors* on the SCRs only (*r*2 = 0.26, *p* = 0.013), with higher SCRs in response to *Odors* on the extremes of the valence spectrum (very unpleasant or very pleasant), but lower responses to (neutral) *Odors* in the middle of the spectrum (**Figure 2B**). These results were confirmed by a supplementary statistical analysis conducted on electrodermal responses to *Odors*. We conducted a repeated measures ANOVA with pleasantness as **three-level** within-subject repeated factor, in which we took into account a third type of neutral *Odors* (two hedonicity scores located around the median score), in addition to pleasant and unpleasant ones. This analysis revealed a main pleasantness effect [*F*(2,34) = 8.31, *p* = 0.001, η2 = 0.33]. A subsequent *post hoc* planned quadratic comparison was performed, with weights of 1, –2, and 1 assigned to pleasant, neutral and unpleasant *Odors*, respectively. This planned comparison was significant [*F*(1,17) = 13.47, *p* = 0.002], confirming that lower SCRs were elicited in response to neutral *Odors* compared to pleasant and unpleasant ones.

In addition, *Odor*-induced heart rate variations correlated negatively with hedonic scores (*r*2 = 0.43, *p <* 0.001; **Figure 2D**). However, no significant correlations with *Fragrance* hedonicity ratings were found for either fragrance-induced SCRs or heart rate variations.

Finally, we examined the effects of stimulus pleasantness on respiratory control measures to rule out any confounds that could cause differences at the physiological level. No significant effects of stimulus pleasantness were found on any of the respiratory control measures [*F*(1,35) = 2.96, *p* = 0.094, n.s., η2 = 0.03, and *F*(1,35) = 0.27, *p* = 0.600, n.s., η2 = 0.01, for abdominal and thoracic respirations, respectively; **Figures 2E,F**], although the general thoracic respiratory amplitude was higher in the *Fragrance* group [*F*(1,35) = 7.52, *p* = 0.001, η2 = 0.18].

#### Facial Muscle Activity

In general, *Odors* elicited a much stronger expressive activity than did *Fragrances* [main group effects: *F*(1,33) = 4.74, *p* = 0.037, η2 = 0.3, and *F*(1,33) = 8.75, *p* = 0.006, η2 = 0.21, for corrugator and zygomaticus, respectively]. We found a significant triple Time × Pleasantness × Group interaction for corrugator activity [*F*(4,132) = 2.45, *p* = 0.050, η2 = 0.07]. In order to examine these effects in more detail, we performed two separate secondary ANOVAs on corrugator activity, where Time (5: corresponding to 5 s × 1 s windows) was introduced as a multiple dependent variable and pleasantness (2) as a within-subject factor for *Odors* and *Fragrances* separately, since muscular activity shows a sequential evolution (see Delplanque et al., 2009).

These analyses revealed a Time × Pleasantness interaction in *Odor*-induced corrugator activity [*F*(4,64) = 2.67, *p* = 0.040, η2 = 0.14], with an increase in the percentage of muscular activity in response to unpleasant *Odors* as compared with pleasant *Odors* in all time windows except the first one (PHC *p*s ≤ 0.004; **Figure 3A**). For better visualization of the effect, the continuous evolution of corrugator activity was plotted both as a function of time and of hedonicity scores. The resulting 3D plot showed a combined slope increasing across time toward lower hedonic values of *Odors* (**Figure 3B**). *Fragrance*induced corrugator activity increased both as a function of unpleasantness [*F*(1,17) = 5.19, *p* = 0.036, η2 = 0.23] and of time [*F*(4,68) = 9.83, *p <* 0.001, η2 = 0.36; **Figure 3C**], although this increase was relatively small compared with that induced by *Odors* (**Figure 3D**).

The zygomaticus also showed increased activity in response to both pleasant olfactory stimuli [main pleasantness effect: *F*(1,33) = 6.50, *p* = 0.016, η2 = 0.16; **Figure 4A**], although the increase in activity over time was more important for *Odors* than for *Fragrances* [Time × Group interaction: *F*(4,132) = 3.94, *p* = 0.005, η2 = 0.11; **Figure 4B**].

# DISCUSSION

In this experiment, we assessed whether subjective, physiological, and expressive indicators differentiate between different ranges of odor and fragrance pleasantness. Our results showed strong distinctions of pleasant and unpleasant *Odors* on the basis of subjective, physiological, and expressive data, in agreement with previous studies (Alaoui-Ismaïli et al., 1997; Bensafi et al., 2002b; Armstrong et al., 2007; Bradley et al., 2008; Delplanque et al., 2009; Sequeira et al., 2009). On the other hand, *Fragrances*, belonging to a more restricted pleasantness spectrum, were mostly differentiated on the basis of their subjective ratings, rather than physiological and expressive indicators.

More specifically, subjective ratings were sensitive to pleasantness, with unpleasant olfactory stimuli perceived as being less familiar and more intense, in line with previous findings (Doty, 1975; Ayabe-Kanamura et al., 1998; Royet et al., 1999; Delplanque et al., 2008), although this distinction between pleasant and unpleasant olfactory stimuli was stronger for *Odors* than for *Fragrances*. At the physiology level, heart rate differentiated between levels of *Odor* pleasantness linearly: the more pleasant the *Odor*, the stronger the decrease, which is in line with previous findings (Soussignan et al., 2005; Delplanque et al., 2009). Electrodermal responses were sensitive to either very pleasant or very unpleasant stimuli. The supplementary analyses performed with an additional category of neutral *Odors* revealed

weaker responses to neutral stimuli, suggesting sensitivity to arousing stimulations, in keeping with the U-shaped relation between odor hedonicity and arousal (Doty, 1975; Bensafi et al., 2002b,c; Winston et al., 2005).

No statistically significant modulation of these two physiological indicators (heart rate, SCR) was observed for *Fragrances*, however, suggesting that the sensitivity of physiological indicators to related odors with a narrow range of pleasantness, such as fragrances, is limited, even although subjective hedonic differentiations were clearly reported by the participants. The fact that no effect of pleasantness was observed in any of the respiratory control measures indicates that it is unlikely that the differences observed at the physiological level could be caused by differential patterns of respiration as a function of odor pleasantness. Finally, pleasantness was also differentiated at the expressive level through corrugator activation and, to a lesser extent, through zygomaticus activation, echoing prior results (Bensafi et al., 2002c; Soussignan et al., 2005; Armstrong et al., 2007; Delplanque et al., 2009). The expressive component responded to both *Odors* and *Fragrances*, although *Fragrance*-related activity was much weaker.

This experiment provided information concerning the ability of classic psychophysiological measures to investigate subtle differences in emotional reaction to olfactory stimuli, as it sheds light on the relation between physiological indicators and subjective ratings when characterizing odors with a wide range of pleasantness versus fragrances with a narrow range of pleasantness. Whereas there were clear differences in physiological reactions to odors that were very different in terms of pleasantness, those differences almost entirely vanished when a particular family of products (i.e., only fragrances) with a restricted range of pleasantness was tested. This does not mean that finding subtle physiological differences in response to a restricted range of products is not possible. Rather, it seems that with classic and easy-to-set-up measures, such subtle differences are unlikely to be observed.

Apart from the technical and methodological constraints, there are clear theoretical reasons to expect such a pattern of results. According to appraisal theories of emotion, e.g., the component process model (Scherer, 1982, 2001), the subjective feeling and the physiological response associated with a specific stimulus (e.g., a given odorant) are separate components whose synchronized modification entails an emotional percept. Although related, subjective feeling and physiological response reflect different components of the emotional response. A modification of the subjective feeling component—which is considered to reflect changes in the other components—will not necessarily entail a difference in the physiological or EMG data, the latter reflecting the expressive component. Our results emphasize the importance of measuring several components of an emotional episode.

On the other hand, the physiological responses observed during an emotional episode should be adapted to the demands of the physical and social environment in order to prepare the individual for action (Frijda, 1987; Sander et al., 2005). Similar to emotional cues triggering adaptive behaviors in reaction to environmental events, olfactory stimuli modulate motivational states in a powerful fashion through their relevance, for example, when malodors induce avoidance reactions through the elicitation of profound aversion or disgust (Royet et al., 2001; Gottfried et al., 2002; Anderson et al., 2003). Olfactory stimuli are thus prone to inducing behavioral adaptations to changes in the environment (Pause et al., 2003), resulting in approach or avoid action tendencies (Frijda, 1987). Olfactory stimuli can even be involved in more complex functions, classified as adaptive behaviors for survival: ingestion, hazard avoidance, social communication, and emotional contagion (see Stevenson, 2010 for a review).

Characterizing consumer preferences by objective physiological and/or EMG measures is a goal that many industries would currently like to attain. These measured responses should be able to differentiate among odors that evoke representations linked to different functions of olfaction (Stevenson, 2010), scattered along a wide pleasantness spectrum. In contrast, it is unlikely that the physiological system would respond differentially when the range of pleasantness is narrow, as is the case with fragrances.

Such subtle differences are well characterized by subjective appreciations, as previous evidence suggested that odor-elicited feelings are complex and varied (Chrea et al., 2009). Aside from the utilitarian functions they embody, odor-borne feelings may also be related to more elaborate forms of hedonic appreciation, such as complex esthetic feelings experienced with music (Zentner et al., 2008; Trost et al., 2012). Odor-borne feelings can be accurately described by specific semantic scales (Chrea et al., 2009; Ferdenzi et al., 2011, 2013; Delplanque et al., 2012), which are a reliable tool for the discrimination of products with similar liking scores such as fragrances or flavored products (Porcherot et al., 2010). In the domain of fragrances, differences in ratings of liking have been found when the same fragrances are rated with or without brand labels (Moskowitz, 1979), an effect commonly observed in food perception (Spinelli et al., 2015). Therefore, an important dimension to consider when it comes to fragrances—in particular, fine perfumes—is luxury because of its ecological occurrence in brand information. A luxurious qualification confers additional value and satisfaction to a given product, as well as supplementary information about its source, yet it may not reflect urgent necessities (Kapferer, 1997; Megehee and Spake, 2012) or differential survival-related functions (e.g., they would all be related to well-being; see Stevenson, 2010). It would thus be interesting to investigate the extent to which self-reported and psychophysiological measures could be influenced by information regarding the luxurious character of a fine perfume. This could be done by presenting the same group of participants with fragrances, with and without the corresponding labels, truthful or not, on different days. Aside from liking, the rewarding sensation experienced during any agreeable sensory stimulation also includes a "wanting" component, which translates into motivation to invest effort in order to obtain such a reward (Berridge and Robinson, 1998; Pool et al., 2015). By measuring, for example, the willingness to pay for a specific product, the wanting component would allow a more complete picture of fragrance-based elicited reward and would perhaps enhance the discriminative power of subjective measures for similarly pleasant products.

# CONCLUSION

In summary, this study shows that emotions elicited by odors that display a wide range of reported pleasantness can be distinguished by both subjective feeling and physiological indicators. These physiological differences almost entirely vanish when odorants belong to a much more restricted pleasantness range, even though the subjective feelings still differ. This work contributes to the literature on emotions by emphasizing the multi-componential nature of emotion and the importance of considering several components when studying olfactoryinduced emotions. Finally, our results address the current trend found in many industries to characterize consumer behavior by using physiological measures. Although differences can be expected in response to heterogeneous products in terms of

# REFERENCES

pleasantness, physiological measures appear to show limited sensitivity in distinguishing among similarly pleasant products.

# FUNDING

This research was supported by the National Center of Competence in Research for the Affective Sciences, financed by a grant from the Swiss National Science Foundation (51NF40- 104897), hosted by the University of Geneva, and also funded by a research grant from Firmenich, SA, to DS and Patrik Vuilleumier.

# ACKNOWLEDGMENTS

The authors thank all the members of the Human Perception and Bioresponses Department of the Research and Development Division of Firmenich, SA, for their precious advice and their theoretical and technical competence.

emotional responses to odors. *Chem. Senses* 34, 49–62. doi: 10.1093/chemse/ bjn052

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Pichon, Coppin, Cayeux, Porcherot, Sander and Delplanque. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Chemosensory Communication of Gender Information: Masculinity Bias in Body Odor Perception and Femininity Bias Introduced by Chemosignals During Social Perception

#### *Smiljana Mutic\*, Eileen M. Moellers, Martin Wiesmann and Jessica Freiherr*

*Diagnostic and Interventional Neuroradiology, Uniklinik RWTH Aachen, Aachen, Germany*

#### *Edited by:*

*Bettina M. Pause, Heinrich Heine University, Germany*

#### *Reviewed by:*

*Jan Havlicek, Charles University in Prague, Czech Republic Katrin Lübke, Heinrich-Heine-Universität Düsseldorf, Germany*

#### *\*Correspondence:*

*Smiljana Mutic<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Emotion Science, a section of the journal Frontiers in Psychology*

*Received: 31 July 2015 Accepted: 10 December 2015 Published: 20 January 2016*

#### *Citation:*

*Mutic S, Moellers EM, Wiesmann M and Freiherr J (2016) Chemosensory Communication of Gender Information: Masculinity Bias in Body Odor Perception and Femininity Bias Introduced by Chemosignals During Social Perception. Front. Psychol. 6:1980. doi: 10.3389/fpsyg.2015.01980*

Human body odor is a source of important social information. In this study, we explore whether the sex of an individual can be established based on smelling axillary odor and whether exposure to male and female odors biases chemosensory and social perception. In a double-blind, pseudo-randomized application, 31 healthy normosmic heterosexual male and female raters were exposed to male and female chemosignals (odor samples of 27 heterosexual donors collected during a cardio workout) and a no odor sample. Recipients rated chemosensory samples on a masculinity-femininity scale and provided intensity, familiarity and pleasantness ratings. Additionally, the modulation of social perception (gender-neutral faces and personality attributes) and affective introspection (mood) by male and female chemosignals was assessed. Male and female axillary odors were rated as rather masculine, regardless of the sex of the donor. As opposed to the masculinity bias in the odor perception, a femininity bias modulating social perception appeared. A facilitated femininity detection in gender-neutral faces and personality attributes in male and female chemosignals appeared. No chemosensory effect on mood of the rater was observed. The results are discussed with regards to the use of male and female chemosignals in affective and social communication.

Keywords: sex, sexual dimorphism, sex recognition, mood, body odor, olfaction

# INTRODUCTION

Humans, although seen as the most highly scented apes (Stoddart, 1990), have been less extensively studied compared to non-primate mammals when it comes to chemosensory communication. Nonetheless, human chemosignalling research has revealed that stable and temporal features of a sender are communicated through the chemical senses (Lübke and Pause, 2015). Especially, when male and female communication via axillary odor is studied, features of a sender affect various levels in a receiver, e.g., social behavior (Frumin et al., 2015), emotional perception (Zhou and Chen, 2009; Albrecht et al., 2011), memory function (Alho et al., 2015), social evaluation (Mitro et al., 2012), attractiveness, and mating preferences (Wedekind and Füri, 1997; Thornhill et al., 2003; Havlicek et al., 2005).

Research on gender-related differences in chemosensory communication reveals the impact of chemosignals on sexual attraction and mate choice (Doty and Cameron, 2009). Chemosensory mate perception is largely affected by female relationship status and menstrual cycle phase (Havlicek et al., 2005; Rantala et al., 2006) as well as intake of hormonal contraception (Roberts et al., 2008). In a study on the chemosensory effect on sexual attraction and mate choice in male raters, it was found that male raters can distinguish between ovulating and non-fertile female body odor (Kuukasjärvi et al., 2004) and that they display higher testosterone levels during exposure to an ovulating female's body odor (Miller and Maner, 2009). Female odor raters explicitly value male body odor pleasantness (Herz and Cahill, 1997; Herz and Inzlicht, 2002) and are able to assess male attractiveness and fluctuating asymmetry, a marker of developmental stability (Thornhill and Gangestad, 1999).

 Nevertheless, body odor sampling studies with both male and female donors are still rare. Mere sex discrimination ability based on female and male axillary odor has been examined in previous research (Russell, 1976; Hold and Schleidt, 1977; Schleidt, 1980; Doty, 1981) stating that humans are able to marginally discriminate between male and female axillary odor. Male body odor is perceived as more musky (Russell, 1976), more intense and less pleasant than female body odor (Hold and Schleidt, 1977; Doty et al., 1978; Schleidt et al., 1981; Mitro et al., 2012). It has further been established that higher chemosensory discrimination of body odors is more frequent for female raters and that the body odor of the opposite sex is expected to smell more pleasant (Hold and Schleidt, 1977; Sergeant, 2010; Mitro et al., 2012).

Ample evidence is pointing to sex-specific differences in male and female body odor. Chemical analyses of volatile compounds in axillary sweat provide information about distinct chromatographic profiles of male and female samples (Penn et al., 2007), and even non-volatile odor precursors of axillary sweat (fatty acids and thiols) were shown to vary concentrationwise in a sex-specific manner (Troccaz et al., 2009). These findings support the idea that sex-related body odor differences do not only exist but can be also communicated among individuals.

Besides natural axillary odor, chemical compounds that are most commonly supposed to have a communicative function are applied to explore chemosensory communication of sex information (e.g., Gustavson et al., 1987; Jacob and McClintock, 2000; Savic et al., 2001; Wysocki and Preti, 2004; Grammer et al., 2005; Lundström et al., 2006; Olsson et al., 2006; Wyart et al., 2007; Zhou et al., 2014) and were shown to affect masculinity and femininity ratings of schematic body movements (Zhou et al., 2014).

Concluding from chemosensory research on sex and gender communication, the conveyed chemosensory information seems to be modulated by the sex of the sender (donor) and the receiver (rater). Taking both factors into account is crucial for an accurate investigation of the still poorly understood chemosensory effect of male and female body odor on higher cognition, emotion, and behavior in a receiver. As it was shown that sex and age of a donor induce rapid mood changes in receivers (e.g., Chen and Haviland-Jones, 1999), affective and social communication via the chemical senses can only be accurately examined in case modulating effects of communicated gender information from the sender to the receiver are known. A study applying the putative chemosignal androstadienone to male and female participants (Hummer and McClintock, 2009) revealed that emotional information processing was altered during its exposure compared to a control odor (clove). While subliminal face processing and perception of emotional words was affected by androstadienone, emotional introspection (mood) was not affected. This finding relates to the discussion by Grammer et al. (2005) of whether chemosignals rather influence socially oriented perception of conspecifics (e.g., evaluation of others, sexual attractant) or self-perception (e.g., as mood enhancer or modulator) in human chemosignalling in general as well as during chemosensory gender communication. This question had not been considered before.

Therefore, we aim to systematically examine the chemosensory information emitted from male and female donors to male and female raters in odor perception, social perception and emotional introspection. We hypothesize that, in a chemosensory rating task, male and female chemosensory samples produce distinguishable intensity and pleasantness ratings. In a masculinity-femininity rating task, we expect male and female chemosensory samples to be correctly assigned by a collective of male and female raters. Furthermore, and beyond mere communication of sex information, we explore whether body odors modulate social or self-perception. As our chemosensory samples convey social information, we expect the perception of social stimuli (in a personality rating task) and conspecifics (in face and word rating tasks) to be modulated rather than introspection (mood rating). More precisely, regarding the rating gender-neutral personality attributes and faces, we expect female chemosensory samples to be associated with a femininity bias and male chemosensory samples to be associated with a masculinity bias.

# MATERIALS AND METHODS

# Participants

The present study was carried out in accordance with the recommendations of the Ethics committee of the medical faculty of RWTH Aachen University and in accordance with the Declaration of Helsinki with written informed consent provided from all participants. In total, 32 healthy participants (raters) took part in the experiment. Participants were healthy, heterosexual (Martins et al., 2005) non-smokers with no current medication or drug intake (Doty and Bromley, 2004).  To ensure that no exogenous odors contaminate the body odors, dietary and hygienic instructions for 2 days prior to the experiment included abstinence from alcohol, caffeine, garlic, onions, spices and the use of deodorants, body fragrances and lotions (Albrecht et al., 2011). Participants showered with scent-free body wash and shampoo, did not shave the armpits and refrained from visiting public pools and saunas. Female participants were scheduled to always participate in the same phase of their menstrual cycle. All females stated to be nonpregnant, did not take hormonal contraception, had experienced a regular menstrual cycle during the six months preceding their participation and were always tested in the same cycle phases each (follicular phase: *n* = 4, periovulatory phase: *n* = 5 and luteal phase: *n* = 7). Phases were defined as a count of post-menstrual onset days based on self-report (Lundström et al., 2006) e.g., for a menstrual cycle length of 28 days, we defined follicular phase from day 1 to 11, periovulatory phase from day 12 to 16 and luteal phase from day 17 to 28. One participant was excluded due to a lack of task compliance. The final sample consisted of *n* = 31 participants (age range = 19–47 years), including 15 males (*M* = 27.80 years, *SD* = 8.83 years) and 16 females (*M* = 29.56 years, *SD* = 9.33 years). The two groups did not differ in age, *t*(29) = 0.539, *p* = 0.594. The odor identification test MONEX-40 (Freiherr et al., 2012) classified all participants as normosmic (*M* = 32.20, *SD* = 2.77; range = 26–38).

# Donation Procedure and Chemosensory Samples

In total, 29 healthy participants (donors) took part in the body odor donation. Female participants did not take hormonal contraception, experienced regular menstrual cycles and stated to be non-pregnant. Donors underwent the same dietary and hygienic instructions as the raters. Two participants were excluded due to acute medication intake prior to the experiment and blood circulation problems. The final sample consisted of *n* = 27 participants (range = 20–49 years), including 14 males (*M* = 25.93 years; *SD* = 8.87 years) and 13 females (*M* = 26.31 years; *SD* = 7.54 years). The two groups did not differ in age, *t*(25) = 0.119, *p* = 0.906.

Upon arrival, participants were informed about the purpose of the study and screened for dietary and hygienic compliance. After cleaning their armpits with scent-free wipes, cotton pads were attached under both armpits. Participants wore a long-sleeved cotton shirt washed with scent-free detergent. In order to increase sweat production, all participants wore a synthetic raincoat. They exercised in a training room with room temperature on an ergometer for 20 min with 100 W/h and 60–80 cycles per minute. After a short break, the pads were replaced and the donation procedure was repeated. Before and after each donation, pulse and blood pressure were assessed (Omron IntelliTM sense, R7 HEM-632-E2; Omron Healthcare Co., Ltd. Kyoto, Japan). To ascertain a correct value, three measurements were performed (immediately after one another) and their mean was used as a final value. This resulted in three measurements in total: before the first 20 min donation, during the break between the first and the second 20 min donation and after the second 20 min donation. Upon successful completion of the donation, the donors were paid 20 Euros.

Immediately after donation, the chemosensory pads underwent an olfactory examination by the experimenter. Pads were not included in case the body odor was not free from perceivable exogenous odors (such as perfume, smoke or spices) or unusual odor intensity was detected (*n* = 2). To minimize odor contamination, pad handling was performed after disinfection of utensils and hands with isopropanol (70%). Each pad was cut in sixteen parts (quadrants of 1 cm × 1 cm). Male and female superdonor pools were created to assure homogeneous odor samples within the experimental groups and to reduce effects of individual variations. This method has been successfully used in prior donation studies (Albrecht et al., 2011; Dalton et al., 2013), which utilized large donor sample groups. Control samples of odorless clean cotton pads (no odor samples without chemosignals) were created and treated like the chemosensory samples in terms of cutting and freezing. The samples were kept in re-sealable storage bags at −80◦C (Lenochova et al., 2009) for no longer than 5 months. Thus bacterial decomposition of the samples was avoided.

# Application Procedure

Raters were invited to three experimental application sessions (within-subject design) within 3 months (one session every 28 days). Females were scheduled to always participate in the same phase of their menstrual cycle. In a double-blind randomized design, participants were exposed to one of the three chemosensory samples (male chemosignals, female chemosignals, and neutral odor) per application session. Thirty minutes before application, quadrants of four donors were randomly chosen from the superdonor pool and put in cotton filter masks. At the beginning and at the end of each of the three application sessions, participants' mood was assessed via selfrating (Watson et al., 1988). The response options were adapted to a 100-point VAS (0 = not at all or very slightly, 25 = a little, 50 = moderately, 75 = quite a bit, 100 = extremely) and mood before and after exposure to the chemosensory samples was compared. After the fitting of the mask under the noses of the participants (Albrecht et al., 2011), a familiarization phase of five minutes was applied to avoid influences of imminent hormonal changes in association with the odor presentation onset that potentially modulate the participants' task performance (Wyart et al., 2007). The experimental tasks took 20–25 min and the odor mask was removed after exactly 30 min of odor exposure. Participants were instructed to breathe normally and rate the masculinity-femininity dimension of the chemosignals, of personality-attributes and of faces. All tasks were computerized. Upon successful completion of all three testing appointments, the raters were paid 45 Euros.

## Odor Perception Tasks

A three-alternative forced-choice test was performed at the beginning of the first session to evaluate odor discrimination capacities. Participants indicated blindly among three samples (two distractors vs. one target sample) the one sample smelling differently with three repetitions of all target and distractor combinations (four discriminations per odor condition and twelve discriminations in total).

At the end of the last session, participants performed an odorrating task where the odor dimensions masculinity-femininity, intensity, and pleasantness were assessed. Hedonic ratings included assessment of intensity, pleasantness, and familiarity of the chemosensory samples (male chemosignals, female chemosignals and no odor sample), and were performed on 100 point VAS ranging from 0 (*not intense/pleasant/familiar at all*) to 100 (*extremely intense/pleasant/familiar*). The masculinityfemininity ratings of the chemosensory samples were performed using 100-point VAS ranging from the endpoint *masculin*e (0), to neutral, (50) to the endpoint *feminine* (100).

#### Social Perception Tasks

For the adjective-rating task, participants rated the masculinity and femininity of 20 gender-neutral adjectives describing persons and personality traits on 100-point VAS.

The gender-neutrality of the personality attributes was identified in a pilot study with 20 male (*n* = 10) and female participants (*n* = 10) evaluating the neutrality of 60 adjectives (Pauly et al., personal communication). This sample included masculinity-related personality attributes (e.g., brutal) as well as femininity-related personality attributes (e.g., caring). The participants rated the gender of the words on a 5-point rating scale with the endpoints very masculine (−2) and very feminine (2). In total, 20 gender-neutrally rated adjectives describing personality attributes (*M* = 0 ± 1 SD) were included in the task (e.g., friendly, childish, discrete).

For the rating of the faces, gender-neutral faces were constructed using the female and male face of the Averaged Karolinska Directed Emotional Faces repertoire (Lundqvist and Litton, 1998) that are not expressing emotions (picture codes: MNES and FNES). Then, hair in both pictures was masked so that only facial features were visible (**Figure 1**). Subsequently, the male and female facial stimuli were merged with three different proportions (40% male + 60% female, 50% each, 60% male + 40% female) using the software MorphX (http://www.norrkross.com/software/morphx/morphx.php). In total, each of those gender-neutral faces was presented five times to the participants.

Other tasks with faces and words were additionally presented during the experimental sessions; the results are discussed elsewhere (Moellers, 2015). As dependent variables, genderneutral personality attributes and faces were rated using 100 point VAS ranging from the endpoint *masculin*e (0), to neutral, (50) to the endpoint *feminine* (100) during application of the chemosensory samples.

# Statistical Analyses

The software package SPSS Statistics 22 (Armonk, NY, USA: IBM Corp.) was utilized for statistical analyses. One-sample *t*-tests were performed to investigate odor discrimination performance. Differences of discrimination performance between the pairs

of chemosensory stimuli (odor pairs: male–female, female–no odor, male–no odor) was assessed with the help of a repeatedmeasures ANOVA with the within-subjects factor odor pair and the between-subject factor sex of the rater.

Normal distribution of the rating data was assessed by onesample Kolmogorov–Smirnov-tests (all *p >* 0.123). Repeatedmeasures ANOVAs with the sex of the rater (male or female) as a between-subject factor and the sex of the donor (chemosensory samples: male chemosignals, female chemosignals, no odor sample) as a within-subject factor were utilized to analyze chemosensory communication of gender information in odor perception and social perception.

Bivariate Pearson correlations were used to assess associations between masculinity-femininity ratings and hedonic ratings (intensity and pleasantness) of the chemosensory samples. Violations of sphericity were adjusted via Greenhouse–Geisser correction and effect sizes were calculated for *F*-tests (η2 p, partial Eta2). Significant main effects and/or interactions were analyzed further using paired-comparison (*t*-tests for two samples and repeated samples) and corrected for multiple comparison using the Bonferroni method. *P*-values *<* 0.050 were considered significant.

# RESULTS

# Donation Exercise Intensity Analysis

In order to investigate general physical fitness, exercise intensity and associated sex differences, systolic and diastolic blood pressure as well as pulse were analyzed. Pulse varied significantly across measurements, *F*(2,48) = 46.271, *p <* 0.001, η2 p = 0.658, but not depending on the sex of the donor, *F*(1,24) = 1.545, *p* = 0.226, η2 p = 0.060. Systolic blood pressure varied significantly across measurements, *F*(2,48) = 14.058, *p <* 0.001, η2 p = 0.369, and depending on the sex of the donor, *F*(1,24) = 5.352, *p* = 0.030, η2 p = 0.182. Diastolic blood pressure varied significantly across measurements, *F*(2,48) = 45.628, *p <* 0.001, η2 p = 0.655, but not depending on the sex of the donor, *F*(1,24) = 1.611, *p* = 0.217, η2 p = 0.063. A significant interaction was found, *F*(2,48) = 65.728, *p* = 0.037, η2 p = 0.128.

No sex differences were found for pulse measures. As blood pressure is generally higher in normotensive men compared to

women (for a review: Lopez-Ruiz et al., 2008), sex differences in systolic and diastolic blood pressure were found prior to donation (**Table 1**). Physical fitness and exercise intensity after donation were comparable across sexes. In order to classify the strength of the physical activity during the donation, the heart rate (pulse in beats per minute; BPM) after the first donation session, male donors: *M* = 104.21, *SD* = 19.42; female donors: *M* = 110.44, *SD* = 15.43, and after the second donation session, male donors: *M* = 107.52, *SD* = 19.84; female donors: *M* = 116.72, *SD* = 16.79, can be classified as moderate and aerobic exercise zones (Fox et al., 1971).

# Masculinity-Femininity Rating of the Chemosensory Samples

Masculinity-femininity rating of body odors varied significantly with sex of the donor (chemosensory samples: male chemosignals, female chemosignals or no odor sample), *F*(2,58) = 9.526, *p <* 0.001, η2 p = 0.247, and the sex of the rater (male or female), *F*(1,29) = 9.866, *p* = 0.004, η2 p = 0.254. Overall, exploratory comparisons revealed more feminine ratings of the no-odor samples, *M* = 54.81, *SD* = 10.78, than both female, *M* = 40.77, *SD* = 15.6); *t*(30) = 3.707, *p <* 0.001, and male chemosignals, *M* = 44.42, *SD* = 13.42; *t*(30) = 3.709, *p <* 0.001. Female raters accurately rated male chemosignals as more masculine than male raters, *t*(29) = 3.599, *p* = 0.001; whereas no sex differences were found for the rating of female chemosignals and no odor samples, all *p >* 0.258; **Figure 2**, **Table 2**.

# Hedonic Ratings of the Chemosensory Samples

Intensity ratings were significantly different across chemosensory conditions, *F*(2,58) = 11.580, *p <* 0.001, η2 p = 0.238. No main effect of the sex of the rater can be reported, *F*(1,29) = 0.003, *p <* 0.958, η2 p = 0.000, but a significant interaction between sex of rater and donor was found, *F*(2,58) = 4.059, *p* = 0.022, η2 p = 0.123. Overall, paired-comparisons revealed no intensity differences between both the male, *M* = 40.26; *SD* = 17.32, and female, *M* = 49.05; *SD* = 18.38, chemosignals, *t*(30) = 2.384, *p* = 0.072, and the male chemosignals and the no odor sample, *M* = 34.19; *SD* = 18.54; *t*(30) = 1.628, *p* = 0.342. Only female chemosignals were perceived to be more intense than the no odor sample, *t*(30) = 5.177, *p <* 0.001. While female raters perceived male chemosignals, *M* = 45.25; *SD* = 15.01, to be as intense as female chemosignals, *M* = 45.06; *SD* = 15.84, *t*(15) = 0.052, *p* = 0.959, male raters perceived male chemosignals, *M* = 34.93; *SD* = 18.49, to be less intense than female chemosignals, *M* = 53.30; *SD* = 20.43; *t*(14) = 3.218, *p* = 0.006, **Figure 3**.

Pleasantness ratings significantly differed depending on the sex of the donor, *F*(1,47) = 11.580, *p <* 0.001, η2 p = 0.285, but not depending on the sex of the rater, *F*(1,29) = 0.148, *p* = 0.704, η2 p = 0.005.

*Pre indicates measurements before physical activity, post indicates blood pressure after both 20 min donation sessions. Pairwise comparisons of sex differences of the donors (independent sample t-test) with Bonferroni-corrected p-values.* ∗*marks significant values p < 0.050.*

TABLE 2 | Mean values and standard deviations, M (SD), for the odor perception task (masculinity-femininity rating) and social perception tasks (gender-neutral personality attributes and faces) by sex of the rater (male and female) and chemosensory sample (no odor, male chemosignals and female chemosignals).

that female chemosignals, *M* = 38.48; *SD* = 17.04, were perceived to be significantly less pleasant compared to male chemosignals, *M* = 50.48, *SD* = 13.08; *t*(30) = 3.492, *p* = 0.006, and compared to the no odor sample, *M* = 52.05, *SD* = 8.89; *t*(30) = 3.945, *p >* 0.001. Higher pleasantness of female chemosignals is associated with higher femininity, *r* = 0.424; *p* = 0.018, and lower intensity ratings, *r* = −0.599, *p <* 0.001. Odor ratings of male chemosignals were not significantly correlated, all *p >* 0.438. Familiarity ratings did not vary with the sex of the donor, *F*(2,58) = 0.210, *p* = 0.811, η2 p = 0.007, or the sex of the rater, *F*(1,29) = 0.338, *p* = 0.565, η2 p = 0.012.

# Discrimination of Chemosensory Samples

During the discrimination task of the odors (twelve trials), the chance level of correct discrimination (33%) equals four correct out of twelve total trials (collapsed across odor conditions) and 1.33 correct out of four trials per odor condition. Participants correctly identified the target odor in 55.89% of all trials, *M* = 6.71, *SD* = 1.95; their general discrimination ability was significantly higher than chance level, one-sample *t*-test: *t*(30) = 7.840, *p <* 0.001. With regards to the different chemosensory conditions, participants were able to discriminate a number of pairs significantly higher than chance level in each odor condition, male-neutral: 58%, *M* = 2.32, *SD* = 0.94; male–female: 47.5%, *M* = 1.90, *SD* = 1.19; female-neutral: 63.75%, *M* = 2.55, *SD* = 0.99; all *t*(30) ≥ 2.68, all *p* ≤ 0.012. Discrimination capacity between the three different odor sample pairs did vary significantly, *F*(2,58) = 3.32, *p* = 0.043. Discrimination between the no odor sample and female chemosignals was significantly better than discrimination between male and female chemosignals, *p* = 0.048. No discrimination differences were found regarding a possible effect of the sex of the rater, *F*(1,29) = 0.063, *p* = 0.804.

### Social Perception

The influence of male and female chemosignals on the perception of gender-neutral personality attributes revealed a main effect of the sex of the donors, *F*(2,58) = 3.967, *p* = 0.024, η2 p = 0.120,

but not of the raters, *F*(1,29) = 0.073, *p* = 0.789, η2 p = 0.003. No difference was found between male and female chemosignals, *t*(30) = 0.385, *p* = 0.703. However, compared to the neutral sample, male and female chemosignals were both associated with more feminine adjective ratings, male: *t*(30) = 2.716, *p* = 0.011; female: *t*(30) = 2.383, *p* = 0.011, **Table 2**.

The influence of male and female chemosignals on the perception of gender-neutral faces (**Figure 4**) did not reveal a main effect of the sex of the donors, *F*(2,58) = 1.685, *p* = 0.194, η2 p = 0.055. However, a significant main effect of the sex of the raters, *F*(1,29) = 27.152, *p <* 0.001, η2 p = 0.484, and a significant interaction of the sex of the donors and raters, *F*(2,58) = 4.892, *p* = 0.011, η2 p = 0.144, was yielded. Paired-comparison revealed that male raters generally rated the faces as more feminine than female raters, all *p* ≤ 0.004. Faces were perceived equally gender-neutral under exposure of female and male chemosignals, *t*(15) = 0.402, *p* = 0.691. While, for male raters, there were no rating differences across chemosensory conditions, all *p* ≥ 0.412, female raters evaluated gender-neutral faces as significantly more feminine (**Table 2**) when exposed to male, *t*(15) = 3.359, *p* = 0.004, and female chemosignals, *t*(15) = 3.010, *p* = 0.009, compared to the no odor sample. Ratings of male and female chemosignals did not differ, *t*(15) = 0.328, *p* = 0.747.

#### Affective Introspection

For the PANAS subscales, no main effects of the sex of the donor or rater were found. Only sample- and sexunspecific stabilizations of the raters' mood were found after the experimental procedure, both for positive mood, *F*(1,28) = 15.220, *p* = 0.001, η2 p = 0.352; pre: *M* = 53.3, *SD* = 13.65; post: *M* = 48.57, *SD* = 15.91, and for negative mood, *F*(1,28) = 4.220, *p* = 0.049, η2 p = 0.131, pre: *M* = 10.89, *SD* = 8.13; post: *M* = 9.63, *SD* = 8.69.

#### Chemosensory Induced Judgment Bias

The judgment bias induced by male and female chemosensory samples on odor perception and social perception is further investigated. *Post hoc*, judgment bias was calculated by subtracting the rating of the no odor sample from the rating of the chemosensory sample (male or female chemosignal). Using this method, a masculinity bias was represented by a negative value and a femininity bias was represented by a positive value. A value not different from 0 represents no bias.

For the main odor perception and social perception tasks (i.e., masculinity-femininity rating of the chemosensory samples, gender-neutral personality attributes and faces rating tasks), judgment bias variables were calculated and chemosensory induced judgment bias was assessed via one sample *t*-tests (test value = 0; **Figure 5**).

For the odor perception task, a masculinity bias induced by male chemosignals was found in female raters, *M* = −15.34, *SD* = 15.77; *t*(15) = −3.893, *p* = 0.001, but not in male raters, *M* = −5.10, *SD* = 14.03; *t*(14) = −1.408, *p* = 0.181. A masculinity bias induced by female chemosignals was found in female raters, *M* = −14.13, *SD* = 24.67; *t*(15) = −2.290, *p* = 0.037, and in male raters, *M* = −13.93, *SD* = 17.32; *t*(14) = −3.116, *p* = 0.008.

The same analysis was computed for the two social perception tasks. For the rating of gender-neutral personality attributes, a femininity bias induced by female chemosignals was found in male raters, *M* = 2.23, *SD* = 2.85; *t*(14) = 3.709, *p* = 0.002, but not in female raters, *M* = 0.75, *SD* = 4.72; *t*(15) = 0.637, *p* = 0.533. A femininity bias induced by male chemosignals was found in female raters, *M* = 1.64, *SD* = 3.01; *t*(15) = 2.182, *p* = 0.045, but not in male raters, *M* = 1.19, *SD* = 2.90; *t*(14) = 1.589, *p* = 0.134.

For the rating of gender-neutral faces, a femininity bias induced by female chemosignals was found in female raters, *M* = 4.44, *SD* = 5.91; *t*(15) = 3.010, *p* = 0.009, but not in male raters, *M* = −1.43, *SD* = 7.26; *t*(14) = −0.763, *p* = 0.548. A femininity bias induced by male chemosignals was found in female raters, *M* = 5.04, *SD* = 6.00; *t*(15) = 3.359, *p* = 0.004, but not in male raters, *M* = −1.14, *SD* = 5.22; *t*(14) = −0.846, *p* = 0.412.

# DISCUSSION

The present study aimed at establishing how humans communicate sex-specific information via body odors involving heterosexual male and female donors and raters. Healthy normosmic males and females were exposed to male and female chemosignals and no odor samples. First, a masculinity bias in human body odor perception was found in a masculinity-femininity rating, hedonic ratings and sensorybased discrimination of the chemosensory samples. Second, human body odor modulated the perception of gender-neutral faces and personality attributes toward a femininity bias.

Concerning the chemosensory communication of sex-specific information via male and female chemosignals in male and female raters, we found that female chemosignals are judged as rather intense and unpleasant by male raters. Although men and women are able to perceive sensory-based differences, the sex of the donor cannot be established from such stimuli. Both men and women seem to judge any body odor as rather masculine.

Our result of a negative correlation between intensity and pleasantness of female body odor (i.e., the more intense, the more unpleasant the perception of female body odor) is in partial accordance with findings of Doty et al. (1978) where inversed pleasantness and intensity ratings were found for female and male axillary odor. This pattern is not restricted to body odor

and was also reported cross-culturally for food and everyday odorants (Distel et al., 1999). Besides the inability to establish masculinity or femininity features based on body odor alone, we conclude that the scent of human body odor seems to be closely associated to the male gender. Two reasons might explain why body odor is perceived as rather masculine. First, perceivable and intense body odor as a consequence of physical activity and strength might cue masculine gender stereotypes of dominance and power. A masculinity bias in sex identification might rely on a semantic tendency of strength being related to masculinity (Doty et al., 1978) – a tendency that might specifically affect female raters presenting higher olfactory abilities. Second, exposure to female body odor might be less frequent than exposure to male body odor. As females purchase and apply fragrances more often and perceive artificial fragrances as more arousing than males (Herz and Cahill, 1997), a diminished exposure to female body odor for both males and females might be the result. Along these lines, a decreased number of opportunities where determining body odor as originating from male or female donors might go along with an inhibited learning process of differentiating female and male body odor. Assuming that – in a social context – the source of a body odor is not clearly identifiable and the scent is less likely to have a female sender, the most adapted response would be to identify the scent as masculine. Another reason for diminished exposure to female body odor might be the decreased intensity compared to male body odor in relation to biological factors. Male body odor is often perceived as more intense and less pleasant than female body odor (Doty et al., 1978; Mitro et al., 2012), an effect that can be related to stronger axillary secretion (Sergeant, 2010) and a higher concentration of sweat-degrading skin bacteria (Jackman and Noble, 1983), steroid hormones, or axillary hair in men. In light of the previous studies and in accordance with our results, we conclude that an unequivocal sex identification based on body odor alone is unlikely to be performed by individuals from industrialized societies.

Investigating the possible modulating effects of chemosensory samples in male and female raters on social perception and selfperception, we aimed to clarify whether sex-specific information in body odors modulate the evaluation of ambiguous conspecifics (personality attributes and faces) rather than modulating affective introspection (mood rating). Supporting the idea that body odors – as social signals – affect the perception of conspecifics rather than introspection, we found that exposure to any body odor induced a femininity bias in social perception. No sex-specific chemosensory effect on a rater's mood was detected. When exposed to any body odor, men and women rated gender-neutral personality attributes as more feminine. Additionally, we found that women perceived gender-neutral faces to be more feminine. Body odors seem to facilitate social cognition and induce a femininity bias, which might be explained by the idea that chemosignals are representatives for social and emotional situations. Evidence arises from neuroimaging studies during which females exposed to chemosignals activate brain areas involved in the assessment of a human quality of chemosensory cues (Zhou and Chen, 2008). Furthermore, conspecifics' body odor is processed in the amygdala and insular regions, unlike other non-human odors (Lundström and Olsson, 2010). Additionally, a strong neural connection of chemosensory processing areas and emotionally relevant limbic areas exists (Albrecht and Wiesmann, 2006). These findings suggest that successful chemosensory communication with a conspecific requires an accurate assessment of emotional cues. Along this train of thought, the femininity bias might be a result activated by emotional sensitivity that is stereotypically associated with feminine referents. As females are more receptive to subtle emotional signals (Pause et al., 2004; Radulescu and Mujica-Parodi, 2013), we find here that the dominant visual signal is modulated more by the chemosensory signal than in male raters.

Taken together, we suspect task-related differences might have led to the sharp contrast of masculinity and femininity biases found. The masculinity bias was established in sex identification via masculinity-femininity ratings and evaluation of the chemosensory samples. The femininity bias was induced by the chemosignals in social perception tasks. Here, two different perceptive and cognitive processes (olfactory perception and evaluation versus multisensory integration and higherorder evaluation) are involved and might explain the opposing findings. While the masculinity bias becomes evident during evaluation of chemosensory information, the femininity bias appears when the olfactory information is a modulating source of information while performing a masculinity-femininity rating on ambiguous visual stimuli. We therefore assume that, besides sex and gender, task complexity might have played an important role on the gender-related biases in chemosensory information transmission.

Addressing the limitations of the present study, we acknowledge methodical limitations in relation to the donation method. Taking into account that different axillary glands are contributing to odorous secretions, we are aware that mainly thermoregulative eccrine glands were stimulated. However, based on knowledge of apocrine and eccrine hyperhidrosis, eccrine gland activity is involved in the transportation of odorous (sebaceous and apocrine) sweat (Hurley, 1989). Also, acknowledging the presence of apoeccrine glands (that are as high in number as apocrine and eccrine glands; Labows et al., 1999), we believe that a stimulation of apocrine and eccrine glands during the experimental set-up resulted in a complex mixture of chemical compounds. Chemosignals were grouped in donor pools characterized by one consistent characteristic: the sex of the donor. The benefit of the superdonor pool in homogenizing across entire group samples, however, represents the inconvenience of being unable to track the individual donor's quadrants that contributed to the chemosensory sample of each receiver. The donation method involving short periods of physical activity was chosen over continuous body odor collection throughout the day for two reasons. First, the entire donation in a laboratory setting assures that no uncontrolled psychological or emotional factors bias the chemosensory samples. Second, to assure that circadian hormonal variations between sexes do not influence the quality of the chemosensory samples (Mong et al., 2011), donation appointment times were kept short and constant for all donors. Additionally, while we controlled for the presence of axillary hair, we are not able to rule out that chemosensory samples might have differed in association with the length of axillary hair (Kohoutová et al., 2012). We find an inverse intensity rating, and we think that the results might be influenced by axillary hair length.

We acknowledge that influences of hormonal contraception were controlled in female donors and raters and influences of menstrual cycle were kept constant in female raters, although not systematically studied. Menstrual cycle differences for female donors were controlled for by using pooled donor sets and, for every female rater, all testing sessions were placed in the same menstrual cycle phase. Female raters in fertile menstrual cycle phases show preferences for symmetrical (Gangestad and Thornhill, 1998) and dominant (Havlicek et al., 2005) body odor of male donors and exhibit an increased chemosensory sensitivity (Doty, 1981). Additionally, only pleasantness and preference ratings were affected by menstrual cycle, while across all cycle phases female raters did not report intensity differences in male body odor (Rantala et al., 2006). Male raters show a preference for ovulatory female body odor (Singh and Bronstad, 2001) only when female donors do not take hormonal contraception (Kuukasjärvi et al., 2004).

Formulating a distinct suggestion on the inclusion of male and female body odor donors in future chemosensory research, we would like to emphasize the importance of the inclusion of female chemosignals when performing chemosensory research on emotional, social, or sexual behavior in humans. To date, male chemosignals are most widely studied in emotional chemosensory communication research, with the argument that their body odor is not affected by menstrual cycle phases. While emotional introspection in a rater does not seem to be

## REFERENCES

affected by the sex of a donor, emotional communication in female raters might still be biased. Observing a masculinity bias in body odor perception and a femininity bias introduced by chemosignals during social perception, we would like to encourage further research to disentangle the influence of cyclic fertility on chemosensory communication of sex and gender information in social perception.

### AUTHOR CONTRIBUTIONS

All authors made substantial contributions to the conception or design of the work. EM performed data acquisition. Data analysis and interpretation was performed by SM, EM, and JF. Drafting of the manuscript was performed by SM. Revising and approving the final version of the manuscript was accomplished by all authors.

### ACKNOWLEDGMENTS

We thank Lies Gysemans, Thomas Vaessen, and Sarah M. Gawronska for their help with data collection and proofreading. This study was supported by a grant from the Interdisciplinary Centre for Clinical Research within the faculty of Medicine at the RWTH Aachen University. This study was conducted as a dissertation project of Eileen Moellers at the Medical Faculty of RWTH Aachen University.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer, Katrin Lübke, and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.

*Copyright © 2016 Mutic, Moellers, Wiesmann and Freiherr. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Chemosensory anxiety cues moderate the experience of social exclusion – an fMRI investigation with Cyberball

#### *Olga A. Wudarczyk1,2\*, Nils Kohn1,2,3, Rene Bergs1,2, Raquel E. Gur4, Bruce Turetsky4, Frank Schneider1,2 and Ute Habel1,2,5*

*1 Department of Psychiatry, Psychotherapy and Psychosomatics, Faculty of Medicine, RWTH Aachen University, Aachen, Germany, 2 JARA – Translational Brain Medicine, RWTH Aachen University and Research Centre Jülich, Aachen, Germany, 3 Department for Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands, 4 Department of Psychiatry, University of Pennsylvania, Philadelphia, PA, USA, 5 Institute of Neuroscience and Medicine (INM-6), Jülich Research Centre, Jülich, Germany*

Recent evidence suggests that the experience of stress can be communicated between individuals via chemosensory cues. Little is known, however, about the impact of these cues on neurophysiological responses during a socially threatening situation. In the current investigation we implemented a widely used paradigm to study social exclusion—Cyberball—to examine whether chemosensory cues signaling anxiety modulate the neuronal effects of ostracism. In a double-blind, within-subjects design, 24 healthy, normosmic participants were presented with chemosensory cues of anxiety (or control samples) and completed the Cyberball task while in a 3T fMRI scanner. Axillary sweat collected from male students awaiting an oral examination served as the anxiety cues while the chemosensory control stimuli consisted of sweat collected from the same individuals participating in an ergometer training session. The neuroimaging data revealed that under the control chemosensory condition, exclusion from Cyberball was associated with significantly higher orbitofrontal cortex and anterior cingulate cortex activity, which is consistent with previous studies in the field. However, when participants were primed with the anxiety sweat, the activity in these regions was not observed. Further, under exposure to anxiety cues during ostracism the participants showed deactivations in brain regions involved in memory (hippocampus), social cognition (middle temporal gyrus, superior temporal gyrus) and processing of salience (inferior frontal gyrus). These results suggest that successful communication of anxiety via the chemosensory domain may moderate the experience of social exclusion. It is possible that the anxiety signals make it easier for the individuals to detach from the

group, pointing to the communicative role of chemosensory anxiety cues in enhancing

Keywords: social exclusion, chemosignals, cyberball, ostracism, olfaction, anxiety

adjustment mechanisms in light of a distressing situation.

#### *Edited by:*

*Géraldine Coppin, University of Geneva, Switzerland*

#### *Reviewed by:*

*Wen Zhou, Institute of Psychology, Chinese Academy of Sciences, China Marilyn Jones-Gotman, McGill University, Canada*

#### *\*Correspondence:*

*Olga A. Wudarczyk, Department of Psychiatry, Psychotherapy and Psychosomatics, Faculty of Medicine, RWTH Aachen University, Pauwelsstrasse 30, 52074 Aachen, Germany<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Cognitive Science, a section of the journal Frontiers in Psychology*

*Received: 11 June 2015 Accepted: 14 September 2015 Published: 09 October 2015*

#### *Citation:*

*Wudarczyk OA, Kohn N, Bergs R, Gur RE, Turetsky B, Schneider F and Habel U (2015) Chemosensory anxiety cues moderate the experience of social exclusion – an fMRI investigation with Cyberball. Front. Psychol. 6:1475. doi: 10.3389/fpsyg.2015.01475*

# Introduction

Recent evidence suggests that the experience of stress can be communicated between individuals via chemosensory cues. It was proposed that upon activation of the sympatheticadrenal medullary (SAM) system, operating closely with the hypothalamus-pituitary-adrenal (HPA) axis, individuals release sweat, which includes physiological markers of anxiety/fear (de Groot et al., 2015). In response to threat, ranging from situations inducing acute fear (such as skydiving, e.g., Mujica-Parodi et al., 2009) to situations inducing acute anxiety (such as anticipation of an oral examination, e.g., Prehn et al., 2006; Prehn-Kristensen et al., 2009), the release of "alarm" signals induces a partial fear state in those exposed to the chemosensory compounds (Prehn et al., 2006; Mujica-Parodi et al., 2009; de Groot et al., 2012). This includes fear-related behavioral and physiological outcomes such as improved cognitive performance (Chen et al., 2006), a bias toward identification of faces as more fearful (e.g., Zhou and Chen, 2009), increased visual field size, and enhanced sensory intake (de Groot et al., 2012), to name just a few. The successful transmission of the "alarm" signals is believed to serve the adaptive function of enhancing sensory vigilance, preparing the organism for environmental dangers (for a review see Stevenson, 2010).

Although current support for the role of chemosensory "alarm" signals in modulating specific emotional, cognitive and physiological processes is abundant (Stevenson, 2010; Pause, 2012), little is known about the impact of these signals on neurophysiological responses during an *actual* threatening context. Given that in everyday life, exposure to chemosignals rarely occurs without a relevant contextual background, it seems critical to assess the impact of chemosensory "alarm" signals during an actual threat. Thus, the current study investigated the impact of chemosensory cues of anxiety on one of the most distressing social situations, i.e., social exclusion. Social exclusion is considered a social danger, as it threatens the basic human need to belong, which is necessary for survival and well-being (Bowlby, 1969; Baumeister and Leary, 1995).

To examine whether chemosensory cues signaling anxiety modulate neuronal effects to social exclusion we first collected axillary sweat from donors in anticipation of an oral examination at a university, which has been linked to experiences of anxiety and the release of emotional chemosignals (e.g., Prehn-Kristensen et al., 2009). We then employed a widely used and well-validated paradigm to study social ostracism in the laboratory environment – Cyberball (Williams et al., 2000; Williams and Jarvis, 2006). In this task participants play a ball-tossing game with two individuals (in reality simulated by the computer) in which, after a series of inclusion trials, they are eventually excluded from the game. The exclusion from Cyberball has been shown to pose a threat to the basic human needs of belonging, feeling in control, maintaining selfesteem, and experiencing a meaningful existence (Williams, 2007; Eisenberger, 2012).

The central question of the current research was whether the communication of anxiety via chemosensory signals modulates the neuronal responses to social exclusion. The evidence suggesting that chemosensory "alarm" cues enhance salience of fear-related socio-emotional cues (Zhou and Chen, 2009) argues for augmented experience of social rejection following the exposure to the anxiety chemosignals. Similarly, studies pointing to emotion contagion via chemosensory "alarm signals" (Prehn et al., 2006; Mujica-Parodi et al., 2009; de Groot et al., 2012) suggest enhanced contagion of anxiety in the context of a distressing situation. Further, the results showing increased fear contagion when olfactory fear is paired with another modality (de Groot et al., 2014) also support this hypothesis. Cumulatively, the expected increased negative experience of social exclusion could be a collective result of chemosensory anxiety and a distressing context. Alternatively, given that chemosensory anxiety cues are potential social threat signals, it cannot be precluded that they are associated with enhancing reappraisal of social rejection to promote fitness. Specifically, from an evolutionary perspective, the successful chemosensory communication of another person's anxiety could be expected to lead to dissociation from the negative experience of social exclusion, in order to enhance productive coping mechanisms during the potentially harmful situation. This hypothesis is supported by an abundance of animal and human studies suggesting a key role for chemosensory "alarm" cues in preparing the organism to tackle a hazardous situation via boosting physiological arousal (Prehn et al., 2006; Inagaki et al., 2008; Pause et al., 2009) and enhancing sensory vigilance (Brown et al., 2004; Chen et al., 2006; Zhou and Chen, 2009; de Groot et al., 2012). These processes may be initiated in preparation to withdraw from the threatening situation (for a review see Pause, 2012).

Taken together, the current study assessed the neuronal implications of exposure to chemosensory anxiety cues in a threatening context of social exclusion. Does the smell of another's person anxiety make us more vulnerable to social exclusion or more prepared to cope with the difficult situation? Increased activity in regions typically involved in processing of social exclusion, including anterior cingulate cortex, medial orbitofrontal cortex, insula, and in regions previously reported to play a role in the processing of socio-emotional information, including the amygdala, hippocampus, and superior temporal gyrus, would support the first answer. By contrast, diminished activity in the regions typically implied in processing of social exclusion would argue for down-regulation of negative feelings associated with ostracism.

# Materials and Methods

#### Ethics Statement

The local ethics committee at the Medical Faculty of RWTH Aachen University approved the current study. The experimental protocol was carried out in accordance with the provisions

of the World Medical Association Declaration of Helsinki. All participants gave written informed consent and were reimbursed for their time.

#### Participants

All volunteers were part of a larger study on the effects of anxiety chemosignals on social cognition. Ten healthy males were recruited as sweat donors. Twenty-four healthy participants (14 men and 10 women) were recruited to take part in the Cyberball study, as sweat recipients. Only males were chosen to donate their sweat, as the apocrine glands in the male underarm area are known to be larger (Doty, 1981). Both genders were included in the fMRI study, as previous research indicated that male stress sweat induces similar neural responses in both gender recipients (Radulescu and Mujica-Parodi, 2013). All Cyberball participants (18–29 years, *M* = 24.33 years, *SD* = 2.91) were screened for fMRI contraindications. In addition, we included only right-handed (Edinburgh Inventory; Oldfield, 1971), non-smoking individuals, who did not suffer from any neurological nor psychiatric illnesses (as assessed via Structured Clinical Interview for DSM-IV, SCID, First et al., 1995), nor showed signs of depression (Beck Depression Inventory; Beck et al., 1961; *M* = 2.9; *SD* = 3.9). Participants' olfactory function, specifically odor identification, was assessed with Monex 40 (Freiherr et al., 2012). According to this task, all participants were normosmic (range: 27–36, *M* = 31.54, *SD* = 2.90).

#### Materials

## Chemosensory Stimuli

The olfactory stimuli consisted of sweat samples collected from males undergoing an important oral examination (anxiety condition) and exercising at a stationery bicycle (sports condition). The sweat donors were invited to take part in the study if they anticipated an important oral examination about which they felt nervous. In addition, their participation was only possible if they reported to be: of Caucasian origin, heterosexual, non-smokers, aged between 18 and 40 yearsold, healthy, physically fit and not taking any medication. Ten males (22–33 years, *M* = 26.40 years; *SD* = 3.75) who fulfilled these criteria were recruited to donate their sweat. They were asked to follow several rules starting 2 days prior to the sweat donation (consistently with, e.g., Zhou and Chen, 2009; de Groot et al., 2012). These included not going into the swimming pool or sauna, not consuming garlic, onion, asparagus, curry, and strongly spiced meals, not drinking alcohol and coffee, sleeping alone, not using deodorant, after-shave, scented creams, and perfumes. In addition, they were asked to use scent-less shampoo and soap provided to them by the experimenter starting 2 days prior to the donation as well as to wash their sheets with an odorless detergent. Before the sweat donation session, they were asked to shower and wear clothes, which they had washed with an odorless detergent provided by the experimenter. All the participants reported following these rules.

The experimental protocol for sweat collection was based on Prehn-Kristensen et al.'s (2009) design. Specifically, the olfactory stimuli were gathered from donors' underarm area with cotton pads attached with plasters for sensitive skin. In the anxiety condition, participants' sweat was collected during anticipation of an important oral examination for 60 min. In addition, at that time, participants' salivary samples were gathered to assess cortisol levels: 60 min before the examination (t0), 30 min before the examination (t1), right before the examination (t2), and right after the examination (t3) using salivettes (Sarstedt, Nuembrecht, Germany). Participants were further asked to complete a Self-Assessment Manikin (Bradley and Lang, 1994) and to evaluate the intensity of six basic emotions (anxiety, joy, surprise, anger, sadness, disgust; Ekman and Friesen, 1975) on visual analog scales, 5 min before the examination. Shortly before the examination the sweat pads were removed. The sports condition consisted of a 10-min introduction to the procedure, three sets of cycling on a stationary bike, 10 min in duration each, with 10 min breaks in between the sets. The salivary samples were collected right at the beginning of the session (t0) as well as 30 (t1), 60 (t2), and 90 min after the start (t3). The Self-Assessment Manikin and Basic Emotions questionnaire were administered after the last bicycle set. Shortly after that the sweat pads were removed. Upon completion of the sweat collection procedure the pads from both sessions were cut into 8 pieces each, blinded by an experimenter not directly involved in this research, sealed in odor-free freezer bags separately per condition and stored at −80◦ Celsius until the day of the examination. The four pieces of the pads per condition, originating from 4 different donors, were defrosted 30 min before the experiment and placed in odorless teabags. During the fMRI experiment, the olfactory stimuli were attached under participants' noses with an odorless strap for the duration of the experimental trial (see *Procedure* for more details). Care was taken to ensure that the chemosensory stimuli did not come into direct contact with participants' skin. Each chemosensory stimulus was prepared and used for one participant only.

#### Cyberball Task

Participants completed the Cyberball task (Williams et al., 2000), a virtual ball-tossing game developed to study social ostracism. The game consists of cartoon images representing other players in the upper corners of the screen and a hand representing the participant at the bottom of the screen, tossing the ball among each other (see **Figure 1A** for illustration). In the current version, the names of the players –"Dieter" and "Nora" – were displayed on the screen next to the animated cartoons. The participants were informed that they were connected to the other two individuals over the Internet, while in reality the game was simulated by the pre-set computer program. The game included two conditions: social inclusion and social exclusion. The participants completed two rounds of the game, one each under exposure to anxiety vs. control sweat cues, presented in a counterbalanced order (see **Figure 1B** for a schematic illustration of the session). For each odor, an inclusion condition was followed by an exclusion condition. Each condition started with a display indicating that the computer was connecting to the other players. The fixation cross was then presented for 15 s, after which the trial began. The participants could toss the ball to one of the two players, by pressing "left" with their index finger or "right"

with their middle finger, on a response box. Each trial was set for 60 throws, with the opponents tossing the ball after 0.5–3.0 s of waiting time (determined randomly). In the inclusion condition, participants played with the two opponents throughout the trial. Each player received the ball on approximately 50% of the throws. In the exclusion condition, the participants received the ball for the first 10 throws, after which they were excluded from the game and did not receive the ball for the rest of the trial (∼50–70 s) while other players tossed the ball among themselves. Following the inclusion condition participants were asked how happy and how angry they were as well as how much they liked the female player and the male player. Following the exclusion condition, participants completed the Need-Threat scale (Williams et al., 2000, 2002) assessing participants' four fundamental needs: belonging (e.g., I felt I belonged to the group), self-esteem (e.g., I felt good about myself), meaningful existence (e.g., I felt nonexistent), and control (e.g., I felt powerful). In order to maintain the cover story, the Need-Threat scale was administered to the participants only after the exclusion condition.

#### Procedure

Before and after the fMRI session participants were asked to evaluate the olfactory stimuli with respect to their valence, intensity and pleasantness. During fMRI scanning, the olfactory stimuli were attached under participants' noses for the duration of the Cyberball run (inclusion condition followed by an exclusion condition plus the accompanying questionnaires). The participants completed two rounds of the game under exposure to the anxiety vs. control sweat cues. The order of olfactory stimuli presentation was counterbalanced across the participants and both the participants and the experimenters were blinded to the nature of the olfactory cues. For a break between the two chemosensory stimulations, the two Cyberball trials were separated by a 10-min anatomical scan, during which no olfactory stimuli were presented. See **Figure 1B** for a schematic illustration of the fMRI session.

#### fMRI Data Acquisition

Functional MRI data were acquired in a three Tesla Tim Trio MR Scanner (Siemens, Erlangen, Germany) at the Department of Psychiatry, Psychotherapy, and Psychosomatics at the Hospital of the RWTH Aachen University. Functional images were collected with an echo-planar imaging (EPI) T2∗-weighted contrast sequence sensitive to blood oxygenation level dependent (BOLD) changes (echo time [TE] = 28 ms, repetition time [TR] = 2 s, flip angle [α]: 77◦, voxel size: 3 mm × 3 mm × 3 mm, 64 × 64 matrix, field of view [FOV]: 192 mm × 192 mm, slice thickness: 3.0 mm, gap: 0.75 mm, number of slices: 34 axial slices, wholebrain, slice acquisition sequence: ascending, 790 volumes per run).

#### Behavioral Data Analyses

The behavioral data were analyzed using IBM SPSS Statistics 20 (SPSS IC, Chicago, IL, USA). The effect sizes are reported according to Cohen's *d* (1988) for paired-sample comparisons and all *post hoc t*-tests.

#### Sweat Donor Physiological and Questionnaire Data

The cortisol levels (in μg/dl) of the sweat donors were extracted at the diagnostic laboratory of the Medical Department of RWTH Aachen University from salivary samples (LDZ: Labordiagnostisches Zentrum Aachen). Three sweat donors were found to produce insufficient salivary amount necessary for the extraction of cortisol values at one of the collection points. The cortisol values of the remaining subjects (*n* = 7) were compared in a 2 (anxiety smell, sports smell) × 4 (time 0, time 1, time 2, time 3) repeated measures analysis of variance (rmANOVA). Follow up comparisons were carried out for each of the salivary sampling points (including the available salivary data for each time point) using Wilcoxon signed-rank test. In addition, the donors' emotional experiences (Basic Emotions and SAM emotions) in the anxiety and sports conditions were compared using Wilcoxon signed-rank test. The Wilcoxon signed-rank test was chosen for these data due to the small sample size of sweat donors (*n* = 10).

#### Odor Differentiation

In order to evaluate whether participants could differentiate the smells with respect to their pleasantness, intensity and valence, the participants' (*n* = 21) ratings of sweat stimuli against these criteria were compared in separate 2 (anxiety smell, sports smell) × 2 (time 1, time 2) × 2 (men, women) rmANOVAs. Three ratings of the participants were not recorded due to measurement errors.

#### Cyberball Behavioral Data

In the Cyberball game, participants' contentment was compared in the exclusion condition versus the inclusion condition (manipulation check) with a paired samples *t*-test, across chemosensory conditions. In addition, participants' evaluation of how much they liked the male and the female opponent in the game, how happy and how angry they felt following the inclusion trial were analyzed with separate 2 (anxiety smell, sports smell) × 2 (men, women) rmANOVAs. Similarly the ratings of the experience of fundamental needs of belonging, self-esteem, control and meaningful existence (NTS; Williams et al., 2000, 2002) following exclusion were analyzed with 2 (anxiety smell, sports smell) × 2 (men, women) rmANOVAs.

#### fMRI Data Analyses

The neuroimaging data were preprocessed and analyzed using SPM8 (Wellcome Trust Center for Neuroimaging1 ) implemented via Matlab 7.10 (MathWorks). Data from two participants who exhibited excessive motion (more than 3 mm in any direction) were excluded from the analyses: the final participant sample for the analyses consisted of 22 participants (12 men and 10 women).

#### Preprocessing

The fMRI data were preprocessed according to standard preprocessing steps (including realignment, coregistration, normalization, and smoothing). The functional scans were first realigned using a two-pass procedure. In this procedure, the first pass—the first scan, and the second pass—the mean scan, were substituted as reference image. Subsequently, the anatomical scans were coregistered to the mean EPI scan. The coregistered images were used for the estimation of spatial normalization parameters using unified segmentation approach (Ashburner and Friston, 2005). The normalization parameters applied to the images transformed them into the standard space as defined by the Montreal Neurological Institute (MNI) and resampled the images to a voxel size of 2 mm × 2 mm × 2 mm. Lastly, smoothing of the images was conducted with a Gaussian kernel of 8mm full-width-at-half-maximum.

#### First Level Analyses

In the first level analyses, the onset and duration vectors for separate Cyberball conditions (i.e., exclusion and inclusion blocks) under the two chemosensory conditions (i.e., anxiety and sports) were convolved with hemodynamic response function (HRF). In addition, the onsets and duration vectors for the sources of noise (i.e., instructions, questionnaires, and waiting time) were modeled out in a separate regressor of no interest. The mean across time for each voxel was modeled by a constant term and low-frequency drifts were removed using a high-pass filter with a cutoff period of 512 s. Temporal correlations were modeled by a first-order regression process as implemented in SPM.

#### Second Level Analyses

Second level analyses were conducted using GLM Flex, (extension to SPM8, see GLM Flex2 ) in which the experimental plan included the following factors: smell (anxiety, sports) and ostracism condition (inclusion, exclusion).

#### Whole Brain Analyses

Whole brain analyses targeted at examining the impact of anxiety chemosensory cues on the experience of social ostracism. We contrasted neural activity during exclusion relative to inclusion separately for: (a) the exposure to chemosensory control cues, (b) chemosensory anxiety cues, and (c) chemosensory anxiety cues – chemosensory control cues. Moreover, we conducted a whole brain smell (anxiety, sports) × condition (inclusion, exclusion) interaction. xJView3 , a viewing program for SPM, was used for exploring and processing of the contrasts and MarsBaR toolbox for SPM4 was used for exploring and processing of the interaction. Additionally, the Anatomy toolbox for SPM (Eickhoff et al., 2005) and the xjView were used for anatomical localization.

#### Volume of Interest Analyses

In order to clarify which condition in our 2 (anxiety smell, sports smell) × 2 (inclusion, exclusion) design, drove the interaction, we identified activation clusters volumes of interest (VOIs) within socio-emotional regions, using MarsBaR toolbox from

1http://www*.*fil*.*ion*.*ucl*.*ac*.*uk/spm

2http://nmr.mgh.harvard.edu/harvardagingbrain/People/AaronSchultz/Aarons\_ Scripts.html

3www*.*alivelearn*.*net/xjview8

4http://marsbar*.*sourceforge*.*net/

all the significantly activated brain regions in the whole brain interaction. The VOIs included the areas previously implied in the Cyberball paradigm (i.e., orbitofrontal cortex and anterior cingulate) as well as the areas involved in social cognition and memory (i.e., superior temporal gyrus, hippocampus, inferior frontal gyrus). Although we are well-aware of the broad involvement of these areas in a wide range of functions, we chose the specified regions based on research (see below), suggesting an intimate relationship with measured processes, of which activation/deactivation patterns in the context of chemosensory anxiety could point to mechanisms by which chemosensory anxiety influences the experience of social exclusion. The VOIs included:

For information about volume, center of mass, peak MNI coordinates, cluster size, and the peak intensity (*T* statistic), refer to **Table 3**.

The mean beta estimates (approximating the activation strength) values in the four clusters were extracted for each subject and each condition against the implicit baseline, and subjected to separate 2 × 2 × 2 rmANOVAs comprising factors: smell (anxiety, sports) × ostracism condition (exclusion, inclusion) × gender (men, women). *Post hoc* analyses were calculated using paired-samples *t*-tests. The comparisons of interest included: anxiety exclusion – anxiety inclusion, sports exclusion – sports inclusion and anxiety exclusion – sports exclusion.

#### Correction for Multiple Comparisons

To correct for multiple comparisons, we applied extent threshold correction as defined by Monte Carlo simulations (3DClustSim; implemented in AFNI; Cox, 1996). This procedure prevents false discoveries resulting from multiple testing. For a threshold at the voxel level of *p* = 0.001 uncorrected, and spatial properties of the current study, 10,000 simulations resulted in an extent threshold of 72 resampled voxels.

## Results

#### Sweat Donors

#### Cortisol

Sweat donors showed higher cortisol levels when awaiting an oral examination than during ergometer training as revealed by a main effect of condition; *F* = 8.774, *p* = 0.025. The Wilcoxon Signed-rank test indicated that there was a significant difference in the cortisol values overall (average rank of 5.50 in the anxiety condition vs. average rank of 0.00 in the sports condition, *Z* = −2.366, *p* = 0.018), as well as specifically at time 0 (average rank of 5.50 vs. 1.00, *Z* = −2.547, *p* = 0.011), time 1 (average rank of 5.50 vs. 0.00, *Z* = −2.805, *p* = 0.005), time 2 (average rank of

TABLE 1 | Descriptive statistics for sweat donors' cortisol levels in the anxiety and sports conditions at times 0, 1, 2, 3, and overall [Mean (SD) and Median].

FIGURE 2 | Sweat donors' emotional responses in the anxiety vs. sports conditions. Sweat donors' evaluations of basic emotions and ratings in Self-Assessment Manikin during anticipation of an oral examination and during ergometer training. The figure depicts participants' mean ratings and standard errors. ∗*p <* 0.05.

4.50 vs. 0.00, *Z* = −2.521, *p* = 0.012), and time 3 (average rank of 6.00 vs. 1.50, *Z* = −2.310, *p* = 0.021). No other main effects nor interactions were observed (all *p >* 0.05). See **Table 1** for an overview of participants' cortisol values.

#### Emotions

The Wilcoxon Signed-rank test revealed that the donors in the anxiety condition showed an increase in reported anxiety as compared to the sports condition (average rank of 5.50 vs. 0.00, *Z* = −2.814, *p* = 0.005). In addition, it revealed a decrease in experienced joy before the examination than during the ergometer training (average rank of 2.50 vs. 6.79, *Z* = −2.053, *p* = 0.040, see **Figure 2** for a visual depiction of mean rating differences). No significant differences in the experience of other basic emotions were observed (all *p >* 0.05). In addition, in the Self-Assessment Manikin, the donors reported feeling less pleasure while awaiting an examination (average rank of 0.00 vs. 5.50, *Z* = −2.680, *p* = 0.007) and more arousal while anticipating

an examination as compared to the training (average rank of 5.50 vs. to 0.00, *Z* = −2.831, *p* = 0.005, see **Figure 2** for visual depiction of mean rating differences). The donors did not report differences in experienced dominance (*p >* 0.05).

#### Olfactory Samples

The participants exposed to the olfactory samples collected from the two situations reported no difference in odor characteristics with regard to pleasantness, *F* = 3.775, *p* = 0.067; intensity, *F* = 2.234, *p* = 0.151, and valence, *F* = 1.055, *p* = 0.317 (see **Figure 3**). No other main effects nor interactions with time nor gender for any of these measures were observed (all *p >* 0.05).

#### Cyberball

#### Manipulation Check

Across chemosensory conditions, the participants reported feeling significantly less contented in the exclusion condition (*M* = 2.46, *SD* = 0.86) as compared to the inclusion condition [*M* = 3.29, *SD* = 0.79; *t*(23) = 3.815, *p* = 0.001, *d* = 1.01, see **Figure 4**].

#### The Experience of Inclusion Under Chemosensory Cues

Following inclusion, under anxiety sweat as compared to sports sweat the participants did not report differences in the experience of happiness (*F* = 0.750, *p* = 0.396), nor anger (*F* = 3.305, *p* = 0.083), nor in how much they liked the male (*F* = 0.013, *p* = 0.909), nor the female participant (*F* = 0.118, *p* = 0.735). Further, the scores were not influenced by gender (all *p >* 0.05).

#### The Experience of Exclusion Under Chemosensory Cues – Need Threat Scale

The chemosensory cues of anxiety did not exert effects on the feeling of belonging (*F* = 1.010, *p* = 0.326), self-esteem (*F* = 0.376, *p* = 0.546), the experience of feeling "in control" (*F* = 3.399, *p* = 0.079), nor meaningful existence (*F* = 0.583, *p* = 0.453, see **Figure 4**). Further, the scores were not influenced by gender (all *p >* 0.05).

evaluation of the experience of basic needs (NTS) following social exclusion. The figure depicts participants' mean ratings and standard errors. ∗*p <* 0.05.

# fMRI

#### Neural Responses to Ostracism Whole Brain Analyses

To assess the effect of chemosensory exposure on neural responses to social exclusion, we examined neural regions that differed in response to social exclusion (compared to inclusion) when participants were exposed to: (a) chemosensory sports (control) cues, (b) chemosensory anxiety cues, (c) chemosensory anxiety cues – chemosensory sports cues. Further, we conducted a smell (anxiety, sports) × condition (exclusion, inclusion) interaction.

#### *Chemosensory sports (control) cues*

We observed increased activity in the clusters encompassing: (1) rectal gyrus, superior orbital gyrus, anterior cingulate, and medial frontal gyrus; (2) anterior cingulate, rectal gyrus, and medial frontal gyrus, (3) superior occipital gyrus, angular gyrus and middle temporal gyrus (for details see **Table 2**; **Figure 5**).

#### *Chemosensory anxiety cues*

No significant suprathreshold activations were observed in the exclusion condition (as compared to inclusion condition).

#### *Chemosensory anxiety cues – chemosensory sports cues*

No significant suprathreshold activations were observed in the exclusion condition (as compared to inclusion condition).

#### *Smell (anxiety, sports)* **×** *condition (exclusion, inclusion) interaction*

Significant activations were observed in 11 clusters. For information about regions within the cluster, hemisphere, volume, center of mass, peak MNI coordinates, cluster size, and peak intensities of the volumes, please see **Table 3** and Supplementary Material for the figure depicting the interaction.

#### VOI Analyses

Four VOIs were selected due to their contextual importance to underlying processes (see methods section for further information) in order to disentangle the smell × ostracism interaction.

#### *Orbitofrontal cortex/anterior cingulate*

A significant main effect of ostracism condition (*F* = 14.913, *p* = 0.001) was identified in the VOI encompassing the superior orbitofrontal cortex and the anterior cingulate (Peak MNI coordinate region: −12, 44, −14, see **Figure 6**), with participants showing increased activity in this region in the exclusion condition (*M* = 0.16, *SD* = 0.25) and relative inhibition in the inclusion condition [*M* = −0.01, *SD* = 0.24, *t*(21) = −3.990,

TABLE 2 | Neural activations in the contrast Exclusion *>* Inclusion for: (a) sports chemosensory condition; (b) anxiety chemosensory condition; (c) anxiety chemosensory condition – sports chemosensory condition.

Anxiety – control Exclusion *>* Inclusion

FIGURE 5 | Sports Exclusion *>* Sports Inclusion (Whole Brain analyses). Significant clusters at *p <* 0.001, with extent threshold = 72 voxels (corresponding to Monte Carlo correction) in the contrast Exclusion *>* Inclusion under the exposure to chemosensory sports (control) cues.

TABLE 3 | Information regarding brain regions, hemisphere, volume, center of mass, peak MNI coordinates, cluster size, and peak intensity (*T* statistic) for significantly activated clusters in the whole brain smell **×** condition interaction.

*The highlighted regions constitute the Volumes of Interest (VOIs) for which beta estimates were extracted for analyses aimed at disentangling the interaction.*

*p* = 0.001, *d* = 0.69]. In addition, a significant smell × condition interaction (*F* = 10.664, *p* = 0.004) revealed that under the chemosensory control cues, the participants showed higher activation in the VOI in the exclusion condition (*M* = 0.26, *SD* = 0.43) than in the inclusion condition [*M* = −0.09, *SD* = 0.38, *t*(21) = 4.278, *p* = 0.000, *d* = *0.*86, see **Figure 6**]. Importantly, the difference in the activity between exclusion and inclusion condition was not found for the anxiety cues [*t*(21) = −0.165, *p* = 0.870]. No other effects were significant (all *p >* 0.05).

#### *Hippocampus*

A significant smell × condition interaction (*F* = 6.786, *p* = 0.017) emerged in the hippocampal region (Peak MNI coordinate: 32, −38, −10, see **Figure 6**). *Post hoc* analyses indicated that under the anxiety smell, the difference between the relative inhibition in the hippocampus in the exclusion condition (*M* = −0.15, *SD* = 0.34), compared to the inclusion condition (*M* = 0.02, *SD* = 0.26) was significant [*t*(21) = −2.206, *p* = 0.039, *d* = 0.56 see **Figure 6**]. Under the control smell, the activity in the hippocampus was enhanced in exclusion (*M* = 0.08, *SD* = 0.33), whereas it was inhibited in the inclusion condition [*M* = −0.07, *SD* = 0.20, *t*(21) = 2.476, *p* = 0.022, *d* = 0.55 see **Figure 6**]. Additionally, in the exclusion condition, there was a significant difference between the inhibition in the hippocampus under the smell of anxiety (*M* = −0.15, *SD* = 0.34) and its activity under the chemosensory control smell [*M* = 0.08, *SD* = 0.33; *t*(21) = −2.075, *p* = 0.05, *d* = 0.69, see **Figure 6**]. The other differences were not significant (all *p >* 0.05).

#### *Middle temporal gyrus/superior temporal gyrus*

A significant smell × condition interaction (*F* = 6.045, *p* = 0.023) and a significant main effect of condition (*F* = 12.505, *p* = 0.002) were observed in the VOI encompassing the right middle temporal gyrus and the superior temporal gyrus (Peak MNI coordinate: 62, −40, 2, see **Figure 6**). As for the main effect of condition, the participants in the exclusion condition showed inhibition in the volume (*M* = −0.04, *SD* = 0.50) whereas in the inclusion condition they showed activity in this region (*M* = 0.24, *SD* = 0.42, *t*(21) = −3.530, *p* = 0.002, *d* = 0.60]. *Post hoc* comparisons disentangling the interaction revealed that under the anxiety smell, the difference between the relative inhibition in the region in the exclusion condition (*M* = −0.32, *SD* = 0.66) as compared to the activity in the inclusion condition (*M* = 0.21, *SD* = 0.46) was significant [*t*(21) = −3.702, *p* = 0.001, *d* = 0.93, see **Figure 6**]. Moreover, in the exclusion condition, there was a significant difference between the relative inhibition in the area under the smell of anxiety (*M* = −0.32, *SD* = 0.66) and the activity under the chemosensory control cues [*M* = 0.24, *SD* = 0.84; *t*(21) = −2.344, *p* = 0.029, *d* = 0.74, see **Figure 6**]. The other differences were not significant (all *p >* 0.05).

#### *Inferior frontal gyrus*

A significant smell × condition interaction (*F* = 11.729, *p* = 0.003) and a significant main effect of condition (*F* = 19.928, *p* = 0.000) were identified in the Inferior Frontal Gyrus (Peak MNI coordinate: −46, 26, 18, see **Figure 6**). Across chemosensory conditions, during exclusion condition the participants showed inhibition in the IFG (*M* = −0.12, *SD* = 0.28) whereas in the inclusion condition they showed activity in this region [*M* = 0.08, *SD* = 0.24, *t*(21) = −4.438, *p* = 0.000, *d* = 0.77]. Under the anxiety smell (but not sports), the difference between the inhibition in IFG in the exclusion condition (*M* = −0.22, *SD* = 0.35) as compared to the activity in the inclusion condition (*M* = 0.15, *SD* = 0.34) was significant [*t*(21) = −5.887, *p* = 0.000,

condition interaction. The significant clusters are at *p <* 0.001, with extent threshold = 72 voxels (corresponding to Monte Carlo correction). ∗*p <* 0.05.

*d* = 1.1, see **Figure 6**]. No other significant differences emerged (all *p >* 0.05).

# Discussion

The central question of the current study was focused on the neuronal implications of chemosensory anxiety signals in the context of social exclusion: do they deepen the negative experience of ostracism or alleviate it? We implemented a widely used paradigm to study social exclusion – Cyberball — while exposing participants to chemosensory cues signaling anxiety (versus control cues). The results revealed that exposure to anxiety cues: (1) modulates the activity in the brain regions involved in processing of negative experience of social exclusion and (2) down-regulates the neuronal areas involved in socioemotional cognition. These results suggest that chemosensory anxiety signals might diminish the experience of social exclusion and facilitate withdrawal from others in the context of a stressful social situation.

#### Neural Responses to Ostracism under Chemosensory Anxiety Cues

Our results extend conclusions of previous studies in the area of chemosensory communication, by showing the association between the exposure to chemosensory anxiety signals and modulation of neural responses during an actual threatening situation of social ostracism. Specifically, during the episode of social rejection the presentation of chemosensory anxiety was not associated with increased activation of the regions previously implied in social rejection (e.g., Eisenberger et al., 2003; Eisenberger, 2012), and also observed in our control chemosensory condition (i.e., anterior cingulate, medial frontal gyrus, orbitofrontal cortices). The lack of activation in these regions, known to be a part of the pain matrix (Eisenberger et al., 2003; Eisenberger, 2012; although a recent meta-analysis by Cacioppo et al., 2013, challenged this perspective arguing that neural correlates of social pain are more complex than claimed by those earlier studies), might imply that chemosensory anxiety moderates the experience of social rejection observed in the control situation.

Moreover, simultaneous deactivations in the brain regions involved in memory (hippocampus), social cognition (middle temporal gyrus, superior temporal gyrus) and salience processing (inferior frontal gyrus) might suggest that successful communication of chemosensory anxiety may be linked to enhancing the preparation of the individual to tackle a stressful episode (e.g., in line with Mujica-Parodi et al., 2009; Zhou and Chen, 2009). Previous studies demonstrated that anxiety signals are driven by the activation of the SAM system (de Groot et al., 2015), which plays a role in the initiation of the fight/flight response. If this state is at least partially communicated to the sweat recipients, it can be presumed that anxiety signals in the context of a distressing social situation may promote the emergence of mechanisms helping to address the hazardous scene e.g., via promotion of distance from the emotional state, or withdrawal from the rejection scenes (also see Koenigsberg et al., 2010; Premkumar, 2012). Accordingly, in the current experiment, upon presentation of the anxiety signals during exclusion, the relative deactivation of the hippocampal area, the region involved in memory, especially encoding processes (Brewer et al., 1998; Wagner et al., 1998), suggests diminished imprinting of negative events into the long term-memory. Given that the opposite pattern was observed in the control chemosensory condition, i.e., rejection led to stronger activity in the region, it appears that chemosensory anxiety plays a role in modulating this process in the context of a challenging situation such as ostracism. Correspondingly, the inhibition of the region encompassing middle temporal gyrus and superior temporal gyrus during the socially distressing episode under anxiety chemosensory cues implies decreased processing of social cues and diminished inclination for "theory of mind" or mentalizing processes, which could be related to increased detachment from the social experience altogether. Similarly, the modulation of inferior frontal gyrus activity in the anxiety (but not in the control chemosensory condition) suggests an influence of anxiety signals on salience processing: while being in a social situation with others (inclusion) the anxiety cues might add salience or arousal to the situation, as the chemosensory input may be perceived as an alarm signal. However, during dissociation from that situation (exclusion), the relative deactivation of the inferior frontal gyrus might be a result of the potential withdrawal from "social threat" signaled by anxiety sweat, which in turn lowers the inferior frontal gyrus activity.

Although, these results might appear counterintuitive in light of findings that anxiety cues enhance the salience of fear-related stimuli and the activation of the socio-emotional regions (Mujica-Parodi et al., 2009; Prehn-Kristensen et al., 2009) they suggest that anxiety signals in an actual, distressing context might override other functions and emphasize the primary role of the chemosensory "alarm" signals in the animal kingdom which is to initiate the organisms' withdrawal behavior from the situation appraised as threatening (e.g., Suh et al., 2004). This interpretation is also in line with a strong body of research showing that the participants exposed to sweat collected from stress-inducing social situation show other signs of preparedness for threat such as improved cognitive performance (Chen et al., 2006), enhanced sensory acquisition (sensory intake, increased visual field size, de Groot et al., 2012) and activation of the withdrawal systems (Prehn et al., 2006; Pause et al., 2009). It should be noted that the withdrawal in light of anxiety signals can be considered a positive outcome, as it facilitates the possible threatening stimulus that leads the sender of the signal to be alarmed in the first place. Future studies should investigate the interaction between the anxiety signals, processing of salience and withdrawal motivation. They should also further decode the mechanisms by which the anxiety signals might promote disconnection from the difficult social situation (e.g., what emotional or physiological tactics are employed by those exposed to the chemosensory cues). This is particularly important, because, given that the currently observed neural regions are involved in a wide range of processes, beyond social cognition and salience, it cannot be precluded, that other processes contribute to the observed results as well.

Lastly, we did not observe any gender effects in the current research, which suggests that the exposure to anxiety signals does not influence neural responses to social exclusion, as a function of gender. Although this is consistent with research showing that both males and females show similar neural activation patterns in response to the olfactory samples of male fear signals (Radulescu and Mujica-Parodi, 2013), studies with larger samples of men and women, designed to test for gender effects specifically are encouraged, to further explore possible differences in experience of social exclusion under influence of chemosensory cues in men and women.

#### Limitations

Although the commonly implemented Cyberball task offers a relatively high ecological validity, it suffers from several limitations when used in the fMRI scanner. Particularly problematic are: (1) the exclusion condition follows the inclusion condition and thus there is a risk of the neural responses being a result of expectancy violation (Somerville et al., 2006), and (2) the length of the blocks leads to a less-than-optimal signal-tonoise ratio. With regards to the current experiment, the choice of a within-subject design, although beneficial for measuring intraindividual variability, might have reduced the experience of social exclusion in the second round of the game (as the repeated rejection trial following inclusion trial appears less realistic). It has to be noted, however, that the order of odor presentation was counterbalanced across participants, such that a "lack of belief in the second exclusion" problem should not occur for different odors. Moreover, several studies have indicated that even a lack of belief in the cover story or knowledge of the exclusion being simulated by the computer is nevertheless associated with the automatic response typical for ostracism (Zadro et al., 2004; Sebastian et al., 2011, also in line with Williams, 2007). Further, the inclusion of a control chemosensory condition, in which non-social cues were presented, would be beneficial to drawing the conclusions regarding the social chemosensory nature of the effects. Lastly, the visual depiction in the differential contrast sports exclusion *>* sports inclusion (**Figure 5**) suggests a possible motion artifact in the third panel. However, given that the motion parameters were included into our design (which should minimize such effects) and the extent of this cluster is in large part in the gray matter (with peak MNI coordinates within Superior Occipital Gyrus), it appears that the activation is not solely artifact related.

#### Conclusion

The current results suggest that successful communication of anxiety via chemosensory domain is associated with downregulation of regions typically implied in the experience of social exclusion during social ostracism. Moreover, it suggests that chemosensory anxiety cues distance individuals from the group, by inhibiting social cognition, salience and memory formation regions during a distressing social event. Cumulatively,

#### References

it is possible that the anxiety signals make it easier for the individuals to withdraw from the hazardous social situation, pointing to the communicative role of chemosensory "alarm" cues in enhancing adjustment mechanisms in light of distressing circumstances.

# Funding

This work was supported by German Research Foundation (DFG, IRTG 1328, International Research Training Group).

# Supplementary Material

The Supplementary Material for this article can be found online at: http://journal*.*frontiersin*.*org/article/10*.*3389/fpsyg*.* 2015*.*01475

an innate avoidance behaviour in Drosophila. *Nature* 431, 854–859. doi: 10.1038/nature02980

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Wudarczyk, Kohn, Bergs, Gur, Turetsky, Schneider and Habel. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# **Effect of fragrance use on discrimination of individual body odor**

*Caroline Allen 1 \*, Jan Havlícek* ˇ *2 and S. Craig Roberts 1*

*1 Division of Psychology, School of Natural Sciences, University of Stirling, Stirling, UK, 2 Department of Zoology, Charles University, Prague, Czech Republic*

Previous research suggests that artificial fragrances may be chosen to complement or enhance an individual's body odor, rather than simply masking it, and that this may create an odor blend with an emergent quality that is perceptually distinguishable from body odor or fragrance alone. From this, it can be predicted that a new emergent odor might be more easily identified than an individual's body odor in isolation. We used a triangle test paradigm to assess whether fragrance affects people's ability to distinguish between individual odors. Six male and six female donors provided axillary odor samples in three conditions (without fragrance, wearing their own fragrance, and wearing an assigned fragrance). In total, 296 female and 131 male participants selected the odd one from three odor samples (two from one donor, one from another; both of the same sex). We found that participants could discriminate between the odors at above chance levels in all three odor conditions. Olfactory identification ability (measured using Sniffin' Sticks) positively predicted discrimination performance, and sex differences in performance were also observed, with female raters being correct more often than men. Success rates were also higher for odors of male donors. Additionally, while performance was above chance in all conditions, individual odor discrimination varied across the three conditions. Discrimination rate was significantly higher in the "no fragrance" condition than either of the fragranced conditions. Importantly, however, discrimination rate was also significantly higher in the "own fragrance" condition than the "assigned fragrance" condition, suggesting that naturally occurring variance in body odor is more preserved when blended with fragrances that people choose for themselves, compared with other fragrances. Our data are consistent with the idea that fragrance choices are influenced by fragrance interactions with an individual's own body odor.

#### **Keywords: deodorant, olfaction, body odor, identification, triangle test, smell**

# **Introduction**