An Assay for Human Chemosignals

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 1 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 373 374 Idan Frumin and Noam Sobel 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 An Assay for Human Chemosignals 375 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 376 Idan Frumin and Noam Sobel 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 An Assay for Human Chemosignals 377 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). 378 Idan Frumin and Noam Sobel 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 <2 mm OD flexible) (see Note 7). 2. Glass Capillaries (50–200 μl) (e.g., Hirschmann Micropipettes or Drummond Microcaps). 3. A rubber bulb or micropipette pump to fit the aforementioned capillaries. 2.4 Preparing Synthetic Putative Chemosignals 1. Synthetic chemosignal, currently commonly used include: (a) Androstadienone (androsta-4,16,-dien-3-one; 4075-07-4) (see Note 8). CAS# (b) Estratetraenol 1150-90-9). CAS# (1,3,5(10),16-estratetraen-3-ol; (c) Androstenone (5α-androst-16-en-3-one ; CAS# 1833916-7) (see Note 9). 2. Diluent—Most commonly propylene glycol (1,2 propanediol; CAS# 57-55-6) (see Note 10). 3. Clove oil/Eugenol (4-Allyl-2-methoxyphenol; CAS# 97-53-0) (see Note 11). 4. Analytical scale. 5. Chemical hood. 6. Glass vials and working tools such as spatula (see Notes 2 and 17). 7. Vortex and/or magnetic stirrer (see Note 12). 2.5 Delivering Chemosignals 1. Band-aids. 2. Pipette. 3. Opaque wide-mouthed jars (ideally glass) with both sealed and mesh covers. 4. Absorbent material, either cotton pads or PTFE beads (see Note 13). 2.6 Measuring Psychophysical and Psychophysiological Responses 1. A computer with stimulus-presentation software, either designated (e.g., Psyscope [43], E-Prime [44]) or programmable (e.g., Matlab [45], LabVIEW [46]). The computer should output to two separate monitors, one in front of the subject chair, and one in the experimenter control room. 2. Pre-rated sets of short emotional movie clips. We use 10-min segments of the nature film “Deep Blue” as “neutral,” 5-min segments of the film “Nine and a half weeks” as “erotic,” a 5-min segment from the movie “The Champ” as “sad,” and 5-min segments from “Monty Python” as “funny” (see Notes 6 and 14). An Assay for Human Chemosignals 379 3. A human approved psychophysiology rig (e.g., AD Instruments [47]) consisting of: 4. 16-channel (or more) biosignal amplifier. 5. Skin conductance (SCR) transducer with two bipolar finger Ag/AgCl Electrodes (surface: 1 cm2). 6. Electrocardiogram transducer (ECG) with three circular Ag/ AgCl conductive adhesive electrodes (0.9 cm diameter). 7. Finger and/or ear pulse transducer (FP/EP) using an IR plethysmograph (size: 15 mm × 15 mm × 6.3 mm). 8. Skin surface temperature transducer (ST) made of a small ceramic-encapsulated metal oxide semiconductor (9.5 mm in length, 2 mm in diameter), designed to operate from 0 to 50 °C. 9. Abdominal and thoracic respiration belt transducers (AR and TR) (30 cm rest length, 10 cm maximum elongation, 4.5 cm in width) mounted on Velcro belts. The transducers contain a piezoelectric device that responds linearly to changes in length (sensitivity, 4.5 ± 1 mV/mm). 10. Motion transducer attached to the subject chair (e.g., highsensitivity (2,500 mV/g) (EGCS, Entran Devices, Fairfield, NJ [48])) (see Note 15). 11. Continuous blood-pressure (BP) finger-cuff monitor (e.g., Finapres Ohmeda 2300 [49]) (see Note 16). 12. Nasal airflow transducer/spirometer and nasal cannula to fit. 13. Biosignal presentation and analysis software (e.g., AD Instruments Chart [47]). 2.7 Measuring Endocrine Responses 1. Bioassy kits for measuring hormones in saliva (e.g., Salimetrics [50] Testosterone and Cortisol kits [51, 52]). 2. Collection tubes (e.g., 15 ml conical PP tubes). Optional— short straws. 3. Refrigerator + Freezer. 4. Polypropylene (PP) Microtubes (e.g., Eppendorf conical 1.5 ml microtubes). 5. Centrifuge (suitable for microtubes (1.5 ml)). 6. Precision pipettes for 20 μl range and for 200 μl range. 7. Multichannel pipette for 200 μl range. 8. Plate agitator/rotator. 9. Spectrophotometric plate reader, to match kit instructions (e.g., for Salimetrics kits—450 nm filter). 10. Computer software capable of 4-parameter sigmoid minus curve fit (4PL logistics non-linear regression) (e.g., SigmaPlot [53], Microsoft Excel Solver tool [54] or MyAssays add-in [55], Matlab [45]). 380 Idan Frumin and Noam Sobel 2.8 Imaging Neural Responses 1. An MRI machine (either 1.5 or 3 T) with gradient capability for functional MRI (fMRI) and appropriate head-coil for functional brain imaging. 2. MRI compatible projection, and MRI compatible earphones. 3. High-powered personal computer with extensive memory and imaging analysis software (e.g., SPM [56], BrainVoyager [57], MR Vista [58]). 3 Methods First, one must select which human chemosignal one intends to study, and collect/prepare it as follows (see Note 17): 3.1 Collecting and Preparing the Chemosignal 3.1.1 Collecting Body-Odor 1. Supply the subject with scentless soap and either a shirt or pads in a sealed plastic bag (see Note 3). 2. Instruct the subject to avoid eating extremely odorous foods that may influence body odor, such as fenugreek, asparagus and garlic. 3. Instruct the subject to wash with the scentless soap before bedtime, not to wear any perfume or deodorant, and wear the supplied shirt over night. 4. In the morning, the subject is asked to put the shirt in the bag and reseal it, and put it in refrigeration for later collection by the experimenter (see Note 4). 3.1.2 Collecting Axillary Sweat 1. Fit the subject with cotton pads placed under the armpits and secured in place using medical adhesive tape. Alternatively, the subject wears a cotton shirt whose armpits are later cut out. 2. Place the subject in the sweat generating condition. Control sweat: Subject is placed on either a treadmill or exercise bicycle for ~30 min. Chemosignal sweat: For the condition of interest, the subject is placed in an appropriate setting. For example, for fear sweat we recommend either tandem skydiving or bungee jumping. Stress sweat can be typically obtained from college students during a statistics exam. 3. Immediately after the sweat is obtained (either control or chemosignal), the relevant emotion is assessed. For example, for fear, after the activity, subjects are requested to rate their level of fear on a 10-point scale ranging from “not afraid at all” to “the most afraid I have ever been.” 4. Immediately after the activity, the pads or the cut armpit areas of the shirt are stored in a sealed plastic bag and kept under refrigeration (see Note 4). An Assay for Human Chemosignals 381 Fig. 3 Collecting tears. To collect tears, the subject watches a sad movie of their own choice in isolation, and once tears start to trickle, they collect them directly into a vial, assisted by a small mirror. Observe the process from neighboring room via one-way mirror or video monitor to assure reliability. 3.1.3 Collecting Tears Emotional Tears and Control Tear collection technique may influence tear fluid content [59, 60]. Ask donors to avoid use of creams, lotions or makeup. Instruct subjects to wash their face without using soap before the collection begins. 1. Seat the subject in a comfortable room, equipped with a display device such as a computer, DVD or projector. 2. Instruct the subject on how to collect tears—by using a mirror and a collection vial, capturing the teardrops directly into the vial as they visibly trickle down the cheek (Fig. 3). 3. To demonstrate this to the subject, use a pipette to apply drops of saline or ringer solution under the subject’s eye, and let the subject capture these drops into the collection vial. This trickled saline will later serve as the control compound. 4. Leave the subject alone in the room, and project the preselected sad movie (see Note 6). Continue to monitor the subject through one-way mirror or video. 5. Ask the subject to call for you once they have obtained tears in the vial, ideally up to the 1 ml mark. 6. Use the tears as soon as possible, but always before 3 h. Trigeminal/Reflexive Tears 1. Seat the subject in a comfortable chair that allows tilting of the head backwards and sideways. 2. An experienced ENT physician should insert a sterilized flexible endoscope into the naris, and make contact with the septum near the inferior turbinate (see Note 7). 3. The contact of the endoscope tip with the trigeminal nerve endings-rich tissue should elicit a tearing reflex in the ipsilateral eye. 382 Idan Frumin and Noam Sobel 4. As soon as tears begin to collect in the eye, tilt the head of the subject gently sideways, while keeping the endoscope in place and avoiding harsh movements. The tilt should be in the direction of the naris—i.e., for a right naris and right eye tilt the head to the right (see Note 18). 5. Use a glass capillary tube to capture the tears as they drop out of the eye (see Note 19). 6. Immediately empty the capillary into a vial. 3.1.4 Synthetic Putative Chemosignals Preparation in Solution The chosen chemosignal can be presented in either solid form or solution (see Note 20). Conduct all the following procedures inside a chemical hood to avoid dust and smell contamination of the surrounding area (see Notes 2 and 17). 1. Weigh the desired amount of solid chemosignal (e.g., for a 2 mM concentration of 10 ml androstadienone solution take ~5 mg solid androstadienone crystals (m.w. 270.4 g/mol) and dissolve in 10 ml of propylene glycol). 2. To generate masked-odor solution (as in [61, 62]), use 1 % w/w eugenol in propylene glycol as the diluent (see Note 11). 3. Rigorously vortex or magnetically stir the solution until no crystals are visible. This might require a couple of hours. Mild heat (<35 °C) can be applied to facilitate dissolution (see Note 12). 4. The above stock solution can be further diluted to any desired concentration by using propylene glycol or propylene glycol + 1 % eugenol (e.g., for 10 ml 250 μM final concentration take 1.25 ml of the above stock solution and mix it with 9.75 ml diluent). For threshold concentrations see ref. [63]. 5. Keep the stock and diluted solutions in an airtight glass vial, preferably under refrigeration and/or well-ventilated storage. 3.2 Narrative of Psychophysiology Experiment The most powerful designs are “within-subjects,” that is, the same subjects are tested in two conditions (chemosignal and control), counter-balanced for order, and double-blind as to compound identity. In other words, neither the participant, nor the experimenter interacting with them, should know which experiment is with the chemosignal or with the control. We recommend that for each subject, the two experiments be conducted day after day and at the same time (see Note 21), so as to minimize external sources of variance. In overview, for each experiment you will obtain a baseline for all measures, then you will conduct a stimulus exposure, and then continue to monitor the response over about an hour. Together, this makes for nearly a 2-h experiment, which you will repeat day after day. An Assay for Human Chemosignals 3.3 Psychophysiology Set-Up 383 1. A same-sex experimenter should greet the subject (i.e., women subject by women experimenter). 2. Obtain informed consent. 3. Escort the subject to the experimental room, and leave them there alone to complete an initial on-screen baseline mood questionnaire. 4. Return to the room, and provide the subject with a collection tube and collect a baseline saliva sample (see Notes 22 and 26). 5. Set the remaining collection tubes, all clearly consecutively numbered, within a holder in easy reach of the subject’s dominant hand. 6. The same-sex experimenter now applies the various psychophysiology transducers to the subject: Skin Conductance Response (SCR): Place the SCR electrodes on the second phalanx of the index and the third digit of the non-dominant hand and attach with Velcro strap. Electrocardiogram (ECG): Paste the two signal electrodes on the left and right lower rib, above the abdomen. Paste the ground electrode on the left ankle. Finger pulse/ear pulse (FP/EP): Using a Velcro strap, place one plethysmograph on the pinky finger of the non-dominant hand. Using an ear-clip, place another plethysmograph on the ear lobe on the side of the non-dominant hand. Skin temperature (ST): Using medical adhesive tape, paste the thermistor directly below the non-dominant axilla. Abdominal and thoracic respiration (AR and TR): Place AR belt around the belt line, and the TR belt around the chest of the subject, just below the axilla. Continuous blood pressure (BP): Place the BP finger-cuff on the third finger of the non-dominant hand, i.e., the finger between the two fingers with SCR electrodes. Spirometer: Fit a nasal cannula to the subject. Motion: This is the only transducer not fitted to the subject, but rather to the armrest of the subject chair. 7. Leave the subject alone in the experimental room, instructing them that if an experimenter later enters, they are not to engage in conversation with them. 8. From the control room, observe that all physiological variables are reading properly at 1 kHz, and if any are particularly noisy, return to the subject room to reconnect sensors as necessary (see Note 23). Start the recording at 1 kHz (make sure you have sufficient memory for a 2-h recording). 384 Idan Frumin and Noam Sobel 3.4 Psychophysiology Experiment 1. Commence with 10-min baseline recording. During these 10 min project a baseline nature film. 2. Project the mood questionnaire. 3. Project instruction to spit into the second collection tube. 4. An opposite sex experimenter now enters the room with a jar containing the stimulus. The experimenter stands next to the subject, opens the jar such that it remains with mesh cover only. At this point, the presentation software should sound an auditory instruction as follows: “At the tone, sniff for the duration of the tone, three, two, one, TONE.” Tone duration should be 1.6 s. At the “two count,” the experimenter should bring the open jar to the nose of the subject, allowing them to take a sniff. The experimenter should pull the jar away at the end of the tone. At this point, consecutive on-screen VAS scales should appear, requesting the subject to rate intensity and pleasantness of the stimulus. The above constitutes one trial. The entire exposure should consist of ten trials, with 30-s inter-trial intervals. 5. After the ten sniffs, the experimenter removes the nasal cannula from the subject, and pastes the pre-prepared stimuluscontaining (~100 μl) band-aid to the upper lip of the subject, to allow continued exposure throughout the experiment (Fig. 4). The experimenter then exits the room. 6. The subject receives an on-screen mood questionnaire. 7. The subject receives on-screen instructions to spit into the third collection tube. Fig. 4 Continued exposure to chemosignals. (a) Use a simple band-aid. (b, c) Fold the band-aid backwards to form a pad with adhesive on the back. (d) Use a pipette to deposit chemosignal onto the pad (~100 μl). (e) Paste the pad on the upper lip An Assay for Human Chemosignals 385 8. Project one of three mood-induction movies (erotic/sad/ funny, counterbalanced for order across subjects). 9. The subject receives an on-screen mood questionnaire. 10. The subject receives on-screen instructions to spit into the fourth collection tube. 11. Project a baseline nature film. 12. Project the second of three movies (erotic/sad/funny). 13. The subject receives an on-screen mood questionnaire. 14. The subject receives on-screen instructions to spit into the fifth collection tube. 15. Project a baseline nature film. 16. Project the third of three movies (erotic/sad/funny). 17. The subject receives an on-screen mood questionnaire. 18. The subject receives on-screen instructions to spit into the sixth collection tube. 19. A same-sex experimenter enters the room, disconnects the subject from the various transducers, and invites them to return at the same time tomorrow. The full timeline for this experiment is in Fig. 5. 20. Freeze saliva samples at or below −20 °C. Samples can be transferred to 1.5 ml microtubes at this stage to save space (see Note 24). 21. On the next day, the procedures are repeated exactly, yet using the second compound (chemosignal/control). The order of the three mood-induction film clips should be counterbalanced across subjects. 3.5 Narrative of Imaging Experiment Those who have an fMRI-compatible olfactometer (likely less than ten labs in the world) can investigate the event-related response to the chemosignal alone. Most labs, however, lacking such an olfactometer, are primarily restricted to examining the brain response to a task or set of stimuli from either the auditory or visual domains, yet under two separate conditions: after exposure to chemosignals Fig. 5 Schematic timeline of psychophysiology experiment. M mood questionnaire, S saliva collection, Set-up hook up all physiological transducers, VN emotionally neutral video clip taken from a nature film, Exp exposure to chemosignal or control, starting with ten sniffs, and then placing of band-aid on upper lip, VE1–VE3 the three mood-induction video clips; sad, happy, and sexually arousing counter balanced for order across subjects 386 Idan Frumin and Noam Sobel versus after exposure to control. We further recommend independent localizer tasks before exposure to either compound. The imaging task can include complex event-related designs, yet here we detail the simplest block-design approach. 3.6 Imaging Experiment 1. Screen subject for MRI compatibility. 2. Place subject in scanner. 3. Obtain full-head T1 weighted high-resolution (1 mm3) 3D acquisition. 4. Obtain a T1 weighted reduced resolution full-brain acquisition at identical parameters (resolution and orientation) to those later used for functional scans. This is for later drawing of regions of interest (ROIs), and realignment. 5. Set parameters for full-brain functional scans (typically T2* weighted). Do not use TRs greater than 2 s, and use low TE to reduce loss in ventral temporal susceptibility regions. 6. Commence block-design localizer: project alternating 30-s movie segments containing either neutral or emotion-ofinterest content. For example, to study sexual behavior related chemosignals, alternate “neutral” and “erotic” clips. To study fear responses, alternate “neutral” and “frightening” clips. Provide 6 alternations across conditions. 7. Extract scanner bed, but instruct subject to remain motionless. 8. With the subject supine in the scanner bed, conduct stimulus exposure as in the psychophysiology experiment. Specifically: 9. An opposite sex experimenter stands next to the subject, opens the jar such that it remains with mesh cover only. At this point, the presentation software should sound an auditory instruction as follows: “At the tone, sniff for the duration of the tone, three, two, one, TONE.” Tone duration should be 1.6 s. At the “two count,” the experimenter should bring the open jar to the nose of the subject, allowing them to take a sniff. The experimenter should pull the jar away at the end of the tone. At this point, consecutive on-screen VAS scales should appear, requesting the subject to rate intensity and pleasantness of the stimulus. The above constitutes one trial. The entire exposure should consist of ten trials, with 30-s inter-trial intervals. 10. After the ten sniffs, the experimenter pastes the pre-prepared stimulus-containing (~100 μl) band-aid to the upper lip of the subject, to allow continued exposure throughout the experiment (Fig. 4). The subject is then reinserted into the scanner. 11. Again obtain a T1 weighted reduced resolution full-brain acquisition exactly as in step #4. 12. Again set parameters for full-brain functional scans exactly as in step #5. An Assay for Human Chemosignals 387 Fig. 6 Schematic timeline of imaging experiment. T1HR high-resolution T1 full head acquisition, T1LR low resolution T1 acquisition at same orientation as later functional scans, T2* functional scans with alternating video clips, “neutral” and “emotion of interest,” Exp extraction from scanner, and exposure to chemosignal or control, starting with ten sniffs, and then placing of band-aid on upper lip 13. Commence block-design experiment exactly as in step #6. 14. Extract subject, and invite them to return at the exact same time on the next day. The full time line for this experiment is in Fig. 6. 15. Repeat all of the above on the next day, yet using the other (chemosignal/control) compound. 3.7 Psychophysiology Analysis Pointers For each subject, for each day, and for each physiological measure, mood question, and hormone in saliva: 1. Extract the baseline values obtained during the 10-min baseline. 2. Extract the values obtained during the ten-sniff stimulus exposure. 3. Extract the values obtained during each type of emotional setting. 4. For each subject, compute a percent change score from baseline for each measure on each day. 5. Compare the change from baseline in the chemosignal condition to the change from baseline in the control condition (see Note 25). 3.8 Endocrine Analysis Pointers 1. Thaw saliva, vortex for a few seconds, and centrifuge at 1,500 × g for 15 min to precipitate particulate material and proteins. Use only the clear supernatant, carefully avoiding disruption of the pellet. 2. Use your specific chosen kit instructions for reagent preparation and needed volumes for analysis. 3. Analysis is best performed in triplicates for each time point of each subject. 4. Arrange the layout of the plate in advance. Leave space as instructed by the kit protocol for standard curve measures and 388 Idan Frumin and Noam Sobel controls, and design the rest of the space so that each triplicate spans two rows and columns, in an L-shaped pattern. This is done in order to avoid intra-column differences across the plate. 5. Upon completion of the kit procedure, you should have a read-out of the plate in relative optical density (OD) values. Use the readouts of the standard curve to fit a 4PL logistics line, using one of the suggested computer programs (see Subheading 2.7, item 10). 6. Average the triplicates and notice any aberrant results. Omit outliers as necessary. 3.9 Imaging Analysis Pointers 1. Conduct the standard pre-processing steps of your analysis scheme (i.e., motion correction, realignment, etc.). 2. Combine the localizer scans (step #6) from Day 1 and 2, and conduct a standard linear contrast to generate functional regions of interest (fROIs). For example, extract areas significantly more or less active in the contrast of “neutral” versus “erotic.” 3. Extract the time course within these fROIs from the later conducted task (step #13). 4. Compute the difference in percent signal change across the two conditions (e.g., “neutral” and “erotic”) within the fROI. 5. Repeat the above for the second day experiment. 6. Compare the change from baseline across the 2 days, chemosignal versus control. 4 Notes 1. The Ekman mood scale originally contains the following 16 variables: Amused; Content; Happy; Calm; Confident; Interested; Angry; Anxious; Annoyed; Bored; Contemptuous; Embarrassed; Stressed; Afraid; Disgusted and Sad. To these we have added a 17th variable: Sexually aroused. To obtain mood state, the 17 variables are displayed in succession with a visualanalogue scale (VAS) ranging between “very” and “not at all,” as in Fig. 1 [22, 64, 65]. 2. If you intend to conduct a single study with human chemosignals, then you can get passed without stainless steel-coated rooms. In contrast, if you are building a lab that will conduct many such studies over time (years), then experimental rooms coated with nonporous material (e.g., Stainless steel, PTFE) are important, as without them you will eventually have contamination across studies. 3. Plastic bags used to store shirts should be odorless, nonabsorbing, non-diffusing and properly sealable. We have found An Assay for Human Chemosignals 389 that aluminum-lined anti-static plastic bags (such as the ones used to store electronic components) work well for this purpose. These are usually sealable using a cheap heat-sealer. Alternatively, zip-lock style sealing is also optional, provided that the seal is hermetic. Amber/Opaque glass jars are also a viable option. 4. Methods for storing chemosignals are critical. In the case of tears, we used only fresh non-refrigerated tears obtained within 3 h or less of their use. Moreover, in a small pilot study we found that storing them for longer periods altered the effects. As to sweat and body-odor, the highest recommended temperature for keeping biological samples is generally 4 °C (regular home fridge temperature). This is a fairly bacteriostatic temperature (i.e., prevents bacterial growth), it slows enzymatic reactions and also reduces evaporation (and hence also loss of volatiles)— but does not allow long-term storage without degradation due to oxidation or eventual bacterial/fungal spoilage. Freezing samples at the usual −20 °C (regular home freezer, but avoid no-frost devices) or −80 °C (laboratory deep freezer) would result in better sample preservation, but care should be given to the rate of freezing: Slow freezing, by simply putting the samples into the freezer chamber, may result in the formation of large crystals of ice. The growing ice crystals might in turn disrupt the membranes of cells found in the sample, resulting in altered composition of the sample due to lost cellular compartmentalization and subsequent sample degradation. Therefore, unless otherwise specified, it is advised to flash-freeze samples prior to long term storage, by transferring the samples into cryogenic tubes and dropping them into liquid nitrogen, followed by storage in freezers. Having said all this, be advised that some plastic materials tend to adsorb lipid components in a way which is aggravated by flash-freezing. Therefore the composition of lipids in the sample might be altered. 5. We use medical grade sterile saline solution (0.9 % w/v Sodium Chloride for IV injection). Alternatives can be Ringer solution (different compositions are available) or a more specialized “artificial tears” (see [66, 67]). 6. To obtain tear donors, we first post an add asking for individuals who cry easily. When they call in, we ask them which film made/ makes them cry, and we obtain this film from the video library. Our experience is that only about 10 % of those who think they cry easily, in fact do so. Such donors, however, can return to lab on a regular basis, and will typically generate 1 ml of tears in response to the same film segment again and again. 7. Flexible thin endoscopes allow the most comfortable means of eliciting reflexive tears. However, other more “low-tech” alternatives, such as a thin long shaft medical transfer swab (e.g., Copan Italia 160C) have also proven useful. 390 Idan Frumin and Noam Sobel 8. We have found that for obtaining good quality steroid chemosignal molecules, Steraloids (Newport, RI) is a good source. 9. Androstenone is a swine pheromone. Pheromones are presumably species-specific. Nevertheless, androstenone has been studied in various human chemosignaling contexts. Like with all steroids, be sure to check purity. 10. While Propylene Glycol (PG; 1,2 propanediol) is a widely used diluting solvent in the odor and fragrance world, Dipropylene Glycol (DPG; 4-Oxa-2,6-heptandiol and 4-Oxa-1,7heptandiol mixture) is also suitable as an alternative. DPG is very similar to PG, although it has lower freezing and higher boiling points, and a lower vapor pressure. Both are almost odorless, and effectively dissolve most moderate to highly hydrophilic compounds and essential oils. Mineral oil can also be considered, as the solubility of steroidal compounds may be higher in hydrophobic solvents, but it is less commonly used. 11. McClintock and colleagues used clove oil to mask the odor of androstadienone [61, 62]. Clove oil is comprised mainly of eugenol, but with varying concentrations (60–95 %, depending on the natural source). To ensure consistent outcome, pure solid eugenol dissolved in the diluent is preferable. Use 1 % w/v eugenol dissolved in propylene glycol. The resulting solution has a recognizable yellow tint, so be sure to use opaque jars for delivery. 12. Dissolution of solid steroid chemosignal compounds might prove hard at higher concentrations (i.e., stock solutions). Possible aids are the use of a sonicator (either ultrasonic bath or probe) and the application of gentle heat (~35 °C) combined with a lengthy stir or vortex. 13. PTFE beads (we usually use 2–3 mm in diameter) are a cheap, inert and simple means of enlarging the effective surface area of solutions. Wet a bed of beads inside a jar to avoid using the liquid solution directly. 14. In different cultures, it may be worth validating these sources in a separate short validation study. For example, American subjects rated clips from “Mr. Bean” as funny, but Israeli subjects did not. In both cultures, however, “The Champ” induced sadness, and “Nine and a half weeks” induced sexual arousal in both sexes. Note that more explicit film clips often induce disgust rather than arousal. 15. The motion sensor is best fitted to the non-dominant hand armrest. This way, the motion trace can later be used to identify motion-artifacts in the physiological data. 16. Finapres Ohmeda 2300 are the best continuous finger-cuff PB monitors, yet they are no longer produced. Thus, each time one shows up on eBay (typically from hospital surplus equipment An Assay for Human Chemosignals 391 dealers, as they were used in child anesthesiology), all the psychophysiologists fight over it. Keep your eyes out for one. 17. We strongly recommend that when working with chemosignals, one applies contamination prevention standards typical of work with radioactive material. Under our working hypothesis of extreme sensitivity to chemosignals, with functional threshold far below conscious detection threshold, the possibility of contaminating a study is ever present. With this notion in mind, it is also advised to work with single-use tools whenever possible (i.e., disposable plastic spatulas, plastic weighing “boats,” etc.). 18. When collecting reflex tears, make sure to tilt the subject’s head in the correct direction, so that tears won’t flow toward the medial canthus and thus drain into the nasolacrimal duct. 19. Hold the glass capillary tube in parallel to the ground or in a small tilt so that the fluid flows freely into the tube aided by gravity and capillary force. Do not over-fill the capillary tube and use the marking to assess the amount of fluid withdrawn. 20. Although many studies have used solid androstadienone crystals as the stimulus, this results in a headspace concentration that is most likely non-biological. 21. Various hormones peak at various times of day. Thus, if you select particular hormones to follow, you should first investigate this phenomenon for the selected hormone, as it may imply particular times of day to avoid, e.g., early morning for testosterone. 22. You should leave a glass of water at reach of the dominant hand of the subject, and suggest that they take a sip after each saliva donation (i.e., ~15 min before their next donation). Otherwise, they might run out of saliva across the 2-h study. 23. A trick to know whether your SCR recording is working is to abruptly bang (once) on the door of the subject room. This should startle the subject, and you should see a clear SCR deviation. 24. Saliva samples should be frozen at −20 °C or below, and not only for long term storage. The freezing itself precipitates proteins (e.g., mucins) out of the saliva liquid, allowing for the separation by centrifugation of the potentially interfering factors for the analysis. However, multiple freeze–thaw cycles should be avoided [51, 52]. 25. Note that this analysis scheme is susceptible to the risks associated with multiple comparisons. This is inherent to casting a wide net. A solution is to first study a small group of subject in a separate pilot study, in order to identify the impact of a given chemosignal. For example, a chemosignal may have profound 392 Idan Frumin and Noam Sobel effects on SCR but not on BP. You should then continue the main study collecting only the measures of interest identified in the pilot. This way you will avoid multiple comparisons. A second alternative is to combine several of the measures into composite measures, as in references [22, 64, 65, 68]. 26. Steroid hormones secretion is episodic in nature. 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