Chapter 27

Chapter 27 An Assay for Human Chemosignals Idan Frumin and Noam Sobel Abstract Like all mammals, humans use chemosignals. Nevertheless, only few such chemosignals have been identified. Here we describe an experimental arrangement that casts a wide net for the possible chemosignaling functions of target molecules. This experimental arrangement can be used in concert with various methods for measuring human behavioral and brain responses, including psychophysiology and brain imaging. Moreover, many of the methodological issues we describe are relevant to any study with human chemosignals. Key words Human, Chemosignals, Pheromones, fMRI, Psychophysiology, Psychophysics

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Introduction All mammals communicate using chemosignals, and humans are no different. Mammalian chemosignaling is especially prominent in reproduction-related behaviors, and this too is true for humans. For example, the clearest case of chemical communication in humans is the phenomenon of menstrual synchrony, whereby women who live in close proximity, such as roommates in dorms, synchronize their menstrual cycle over time [1]. This effect is mediated by an odor in sweat. This was verified in a series of studies where experimenters obtained underarm sweat extracts from donor women during either the ovulatory or follicular menstrual phase. These extracts were then deposited on the upper lips of recipient women, where follicular sweat accelerated ovulation, and ovulatory sweat delayed it [2, 3]. Moreover, variation in menstrual timing can be increased by the odor of other lactating women [4], or regulated by the odor of male hormones [5, 6]. A second human reproduction-related chemosignaling behavior relates to mate selection. The human genome includes a region called Human Leukocyte Antigen (HLA), or more broadly termed Major Histocompatibility Complex (MHC), which consists of many genes related to the immune system, in addition to olfactory

Kazushige Touhara (ed.), Pheromone Signaling: Methods and Protocols, Methods in Molecular Biology, vol. 1068, DOI 10.1007/978-1-62703-619-1_27, © Springer Science+Business Media, LLC 2013

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receptor genes and pseudogenes. Several studies have found that women can use smell to discriminate between men as a function of similarity between their own, and the men’s HLA alleles [7–11]. The “ideal” smell of genetic makeup remains controversial, yet most evidence suggests that women prefer an odor of a man with HLA alleles not identical to their own, but at the same time not too different [11, 12]. In turn, this preference may be for MHC heterozygosity rather than dissimilarity [13]. This chemosignaling dependent mate preference is plastic. For example, single women preferred odors of MHC-similar men, while women in relationships preferred odors of MHC-dissimilar men [14]. Moreover, olfactory mate preferences are influenced by the menstrual cycle [15–18] and by hormone-based contraceptives [7, 8, 19]. Finally, olfactory influences on mate preferences are not restricted to women. Men can detect an HLA odor different from their own when taken from either men or women odor donors, and rate the similar odor as more pleasant for both of the sexes [8, 13]. In addition, men preferred the scent of common over rare MHC alleles [13]. Moreover, unrelated to HLA similarity, male raters can detect the menstrual phase of female body odor donors. The follicular phase is rated as more pleasant and sexy than the luteal phase [18], an effect that is diminished when the women use hormonal contraceptives [13, 20]. These behavioral results are echoed in hormone expression. Men exposed to the scent of an ovulating woman subsequently displayed higher levels of testosterone than did men exposed to the scent of a non-ovulating woman or a control scent [21]. Moreover, a recent study on chemosignals in human tears revealed a host of influences on sexual arousal [22]. Sniffing negative-emotionrelated odorless tears obtained from women donors induced reductions in sexual appeal attributed by men to pictures of women’s faces. Sniffing tears also reduced self-rated sexual arousal, reduced physiological measures of arousal, and reduced levels of testosterone (recently also seen by Oh and colleagues [23]). Finally, functional magnetic resonance imaging revealed that sniffing women’s tears selectively reduced activity in brain substrates of sexual arousal in men [22]. Human chemosignaling is not restricted to reproductionrelated behavior. Although many types of social chemosignaling have been examined [24], here we will detail one particular case, and that is the ability of humans to smell fear. Fear or distress chemosignals are prevalent throughout animal species [25, 26]. In an initial study in humans, Chen and Haviland-Jones [27] collected underarm odors on gauze pads from young women and men after they watched funny or frightening movies. They later asked other women and men to determine by smell, which was the odor of people when they were “happy” or “afraid.” Women correctly identified happiness in men and women, and fear in men. Men

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correctly identified happiness in women and fear in men. A similar result was later obtained in a study that examined women only [28]. Moreover, women had improved performance in a cognitive verbal task after smelling fear sweat versus neutral sweat [29], and the smell of fearful sweat biased women toward interpreting ambiguous expressions as more fearful, but had no effect when the facial emotion was more discernible [30]. Also, subjects had an increased startle reflex when exposed to anxiety-related sweat versus sportsrelated sweat [31]. Finally, imaging studies have revealed dissociable brain representations after smelling anxiety sweat versus sports-related sweat [32]. These differences are particularly pronounced in the amygdala, a brain substrate common to olfaction, fear responses, and emotional regulation of behavior [33]. Taken together, this body of research strongly suggests that humans can discriminate the scent of fear from other body odors, and it is not unlikely that this influences behavior. How can we assay whether a given substance is, or contains, a human chemosignal? The rational for how to do this is simple: Once we have identified a chemosignal and a behavior we think it relates to, we can measure that behavior, or its neural substrates, with and without exposure to the chemosignal. Thus, if we have identified a fear-related chemosignal, we can measure fear in the presence of the chemosignal versus the presence of an unrelated control substance, or measure brain activity in the amygdala for example, again in the presence of the chemosignal versus the presence of an unrelated control substance. However, in many cases we may have a potential chemosignal in hand without a clear notion regarding its expected influence. With this in mind, we have developed a behavioral assay that provides a rather widely cast net. This is an experiment aimed at probing for a host of potential psychological, physiological, and brain responses. In this chapter we will describe this assay. The human responses within this assay can be measured with several standard methods, for example psychophysiology and brain imaging. The general application and analysis of psychophysiology and brain imaging has been detailed in various chapters of this series [34–37], and is indeed beyond the scope of one text. Therefore, here we will concentrate on the unique aspects of assaying human chemosignals with these methods. To reiterate, this chapter is not intended to teach psychophysiology and brain imaging, but rather how to bring human chemosignals into this environment. We also detail various alternatives regarding major design aspects of such experiments. For example, for stimuli one can use the full human-derived media that presumably contains the chemosignals (sweat, tears, etc.), or individual synthetic molecules that have been dubbed putative human chemosignals. Also, one can measure the direct impact of the stimuli, or the influence of the stimuli on some task, such as emotional appraisal, or startle response. An additional major design aspect with extreme alternatives has to

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do with stimulus delivery. If one wants to deliver the stimuli with millisecond temporal resolution, one needs an olfactometer. These devices, which can be self-built [38–40] or bought [41], are complicated and expensive, and are therefore in the hands of relatively few labs. With this in mind, in this chapter we will restrict our description to methods that do not call for an olfactometer. Finally, Subheading 4 contains various insights from our experience with apparently small decisions that sometimes make all the difference between a successful and unsuccessful experiment. We wish you good luck with yours.

2 2.1

Materials General

1. Ethical approval for the procedures from appropriate authorities (Helsinki or IRB committee). 2. Human volunteers (~30 per study). 3. General questionnaires. These should include a comprehensive demographics questionnaire, and the Ekman mood questionnaire [42] (see Note 1). Questionnaires should be made executable on-screen using presentation software (Fig. 1). 4. Well-ventilated room, subserved by Carbon and HEPA filtration, ideally coated with odorant non-adherent material such as stainless steel (see Note 2). The room should be observable from a neighboring control room through one-way mirror and/or video monitors such that subjects can be left alone in the room during the experiments. An intercom between experimental and neighboring control room is helpful. 5. A subject-chair that is both comfortable and adjustable, ideally a dentist-type patient chair (Fig. 2). The chair should be equipped with a wide armrest that can be refitted for either the left or right arm. This armrest is for the non-dominant hand

Fig. 1 Schematic of on-screen Visual Analog Scale (VAS). This graphic is presented on the screen in front of the subject. The subject uses the mouse to drag the marker horizontally to a position that reflects their self-assessment of the current mood in question (in this case “happy”). Once the marker is in the appropriate place, the subject clicks the mouse to enter their judgment, and the next mood question appears (e.g., “happy” is replaced with “sad”). This continues for the 17 mood questions (see Note 1). Although this may seem crude, it is in fact informative and reliable

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Fig. 2 Subject set-up in chair. Subject comfortably seated in stainless steel room. Visible transducers include body temperature (temp.), ear pulse (EP), nasal respiration (Cannula), thoracic respiration belt (TR), abdominal respiration belt (AR), and inset highlights skin conductance sensors (SCR), finger pulse (FP), and blood pressure (BP). Note monitor in easy viewing angle, and one-way mirror behind monitor, which allows viewing from neighboring experimenter control room

later fitted with physiological transducers. The opposing armrest should have a keyboard and mouse holder, to allow subject responses. Display hardware, e.g., computer monitor, should ideally be situated in comfortable viewing angle from the chair. 2.2 Collecting Body-Odor or Sweat

1. Scentless soap. 2. Cotton pads or cotton shirts. 3. Medical adhesive tape. 4. Sealable aluminum-lined plastic bags (sized to contain aforementioned pads/shirts) (see Note 3). 5. Refrigeration for the samples at 4 °C or below (see Note 4). 6. For sweat: An emotional setting for the active condition (see Subheading 3.1.2), and a treadmill/exercise bicycle for the control.

2.3

Collecting Tears

In all cases: 1. Vials/Tubes (preferably glass, wide opening (1–2 cm diameter)). 2. Saline/physiological solution, or ringer solution (see Note 5).

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2.3.1 Emotional Tears

1. A computer or TV/DVD set for screening movies (headphones optional). 2. A sad movie (ideally chosen by the subject) (see Note 6). 3. A small make-up mirror, or similar.

2.3.2 Trigeminal/ Reflexive Tears

1. Nasal endoscope (ideally