ORIGINAL RESEARCH
published: 22 April 2020
doi: 10.3389/fnins.2020.00255

An Evolutionarily Threat-Relevant
Odor Strengthens Human Fear
MemoryJessica E. Taylor 1* , Hakwan Lau 1,2,3 , Ben Seymour 4,5,6 , Aya Nakae 7 , Hidenobu Sumioka 8 ,Mitsuo Kawato 1,4 and Ai Koizumi 1,4,9*
1

Department of Decoded Neurofeedback (DecNef), Computational Neuroscience Laboratories, Advanced
Telecommunications Research Institute International, Kyoto, Japan, 2 Department of Psychology, Brain Research Institute,
University of California, Los Angeles, Los Angeles, CA, United States, 3 Department of Psychology, University of Hong Kong,
Pokfulam, Hong Kong, 4 Center for Information and Neural Networks (CiNet), National Institute of Information andCommunications Technology (NICT), Osaka, Japan, 5 Computational and Biological Learning Lab, Department ofEngineering, University of Cambridge, Cambridge, United Kingdom, 6 Department of Neural Computation for
Decision-Making, Cognitive Mechanisms Laboratories, Advanced Telecommunications Research Institute International,
Kyoto, Japan, 7 Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan, 8 Hiroshi Ishiguro Laboratories,
Advanced Telecommunications Research Institute International, Kyoto, Japan, 9 Sony Computer Science Laboratories, Inc.,Tokyo, Japan

Edited by:Kathrin Ohla,
Institute of Neuroscience andMedicine (FZJ), Germany
Reviewed by:
Valentina Parma,
Temple University, United States
Markus Fendt,
University Hospital Magdeburg,
Germany
Yuri Masaoka,
Showa University, Japan*Correspondence:
Jessica E. Taylor
jessie.elizabeth.taylor@gmail.com
Ai Koizumi
bellkoizumi@gmail.com
Specialty section:
This article was submitted toPerception Science,a section of the journal
Frontiers in Neuroscience
Received: 06 November 2019
Accepted: 06 March 2020
Published: 22 April 2020
Citation:
Taylor JE, Lau H, Seymour B,
Nakae A, Sumioka H, Kawato M and
Koizumi A (2020) An Evolutionarily
Threat-Relevant Odor Strengthens
Human Fear Memory.
Front. Neurosci. 14:255.
doi: 10.3389/fnins.2020.00255

Olfaction is an evolutionary ancient sense, but it remains unclear to what extent it can
influence routine human behavior. We examined whether a threat-relevant predator odor
(2-methyl-2-thiazoline) would contextually enhance the formation of human fear memory
associations. Participants who learned to associate visual stimuli with electric shock inthis predator odor context later showed stronger fear responses to the visual stimuli thanparticipants who learned in an aversiveness-matched control odor context. This effectgeneralized to testing in another odor context, even after extinction training. Resultsof a separate experiment indicate that a possible biological mechanism for this effectmay be increased cortisol levels in a predator odor context. These results suggest that
innate olfactory processes can play an important role in human fear learning. Modulatory
influences of odor contexts may partly explain the sometimes maladaptive persistenceof human fear memory, e.g., in post-traumatic stress disorders.
Keywords: fear memory, human olfaction, predator odor, innate fear, contextual memory modulation

INTRODUCTION
Although odors can act as powerful cues for triggering recall of fear memories (Vermetten andBremner, 2003; Hinton et al., 2004), their ability to act as contextual modulators of fear memory
formation is less well understood. Many previous studies have exposed experimental animals toodors at the time of fear memory formation; however, these studies typically used odors as a directcue for the learned association. Often, in these experiments, neutral odors were used as conditioned
stimuli (CS) (e.g., Richardson et al., 1999; Paschall and Davis, 2002; Sevelinges et al., 2004; Joneset al., 2005; Kass et al., 2013; Shionoya et al., 2013) and/or aversive odors were used as unconditioned
stimuli (US) (e.g., Blanchard et al., 2001, 2003; Endres and Fendt, 2007). The number of studies
exposing human subjects to odors at the time of fear or negative memory formation is far fewer,
but these have also tended to use odors as a direct cue for the learned association (either as a CS,
e.g., Kirk-Smith et al., 1983; Epple and Herz, 1999; Parma et al., 2015; or as a US, e.g., Schneider
et al., 1999; Hermann et al., 2002). Therefore, despite suggestions that their pervasiveness mightcause odors to provide particularly strong contextual information (Herz and Engen, 2010), there

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remains little known about how an odor might affect fear
learning when it serves (neither as CS or US but) simply
as a background context. Better elucidation of modulatory
contextual features could be beneficial for the understandingof why some, but not all, humans who experience trauma
develop robust fear memories that potentially lead to fearrelated disorders.
In this study, we hypothesized that an odor providing
evolutionary threat-relevant contextual information may
influence human fear memory formation. This is because
humans display threat-related psychological responses in
evolutionary threat-relevant odor contexts (Stevenson, 2010;
Wisman and Shrira, 2015), indicating that we are capable ofprocessing their threat relevance. For example, putrescine(the smell of decay) has been shown to elicit human threat
management mechanisms (Wisman and Shrira, 2015), andthe body odor of scared humans has been shown to influence
the cognitive performance of other humans (Chen et al.,2006). A review of the animal literature shows that a simple
context containing a predator odor is often sufficient to induce
evolutionary fear-like responses (Apfelbach et al., 2005). The
mechanism behind the effect of a predator odor is thought
to be innate (Kobayakawa et al., 2007) and has been found
to exceed the effect of an experimentally fear-conditioned
threat odor on appetitive behavior (Isosaka et al., 2015). The
molecular and neural mechanisms behind innate fear-like
responses driven by predator odors are relatively well known(Kobayakawa et al., 2007; Isosaka et al., 2015; Wang et al.,2018). If predator odors affect humans innately as well, then thisopens the scope for a broad range of future research. Therefore,
in this study, we decided to specifically test the effect of apredator odor.
Predator odors may not typically be thought to be innately
threat-relevant to humans, but they have previously beensuggested to contain chemicals that have been reliable indicatorsof predators in our ancestral past (Stevenson, 2010). Our fear
circuits have evolved over history so that we potentially retain the
innate capacity to process certain stimuli from our evolutionary
past as threatening, even if they no longer signify real danger
to us (LeDoux, 2019). Indeed, it has been previously suggested
that humans are innately prepared to defend themselves against
the visual cues of predators (Ohman and Mineka, 2001) and,given that one of the major functions of the human olfactory
system has been proposed to be threat detection based on
evolutionary experience (Stevenson, 2010), we may likewise
have preparedness toward their odors. In humans, a potent
analog of a sulfur-based predator odor [2-methyl-2-thiazoline(2MT)] (Kobayakawa and Kobayakawa, 2011) has been reported
to induce changes in heart rate (R. Kobayakawa, personalcommunication, September 5, 2007). This odorant has beenshown to work on a chemoreceptor in mice (transient receptor
potential ankyrin 1; Wang et al., 2018), which is also present
in humans. Therefore, in the current study, we used 2MT asa background context while human participants learned fearassociations between other stimuli. The aim was to test the
hypothesis that human fear learning may be modulated byevolutionary ancient olfactory mechanisms.

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MATERIALS AND METHODS FOR MAIN
EXPERIMENT
Ethics Statement
This study was approved by the Ethics Committee of the Review
Board of Advanced Telecommunications Research InstituteInternational, Japan, and all experiments were performedin accordance with relevant guidelines and regulations.
All participants provided written informed consent prior
to participation.

ParticipantsParticipants were 40 Japanese females with normal or corrected tonormal vision. Only female participants were recruited to exclude
the potentially confounding factor of sex on conditioning (Dalla
and Shors, 2009; Inslicht et al., 2013) and to reliably measure skinconductance, which is often found to be more reactive amongfemales (Eisdorfer et al., 1980; Chentsova-Dutton and Tsai, 2007).
All participants were right-handed. Each participant was paid
U5,000 per day for 5 consecutive days, with a bonus U5,000
on the last day (U30,000 in total). Participants were randomly
assigned to complete conditioning in the 2MT context (2MT
group) or in the control odor context (Control group). The
data of four participants (two from each group) were excluded
from all analyses. This was due to technical issues with theskin conductance response (SCR) electrodes during the Test
phase of the experiment, which was the phase of most interest.
There remained 18 participants in each group. There was no
significant difference in age between groups [for the 2MT group:M = 26.5 years, SD = 4.4; for the Control group: M = 25.6 years,
SD = 4.1; t(34) = 0.63, p = 0.532].
The considerable time required to run each participant in
the main experiment (1.5 h per day × 5 days per participant)necessitated a modest sample size (20 participants per group × 2
groups). A simple post hoc power analysis [for a betweengroup independent-samples t-test examining average SCRs to
CS+ s (minus those to the CS−) from the Test phase of themain experiment] revealed relatively high power (1- β error
probability = 0.96). We therefore believe that the sample size usedin the main experiment was sufficient and appropriate for our
experimental design and hypotheses.

Materials
Stimulus Presentation
Visual stimuli were presented on an LCD display using thepsychophysics toolbox (Brainard, 1997) running on Matlab 7.1(MathWorks, Inc.).

Physiological Measurements and Electric Stimulation
SCR was recorded via BrainVisionRecorder using BrainAmp(Brain Products GmbH, Germany) Ag/AgCl sintered electrodes.
Electric shocks were administered via electrodes that wereconnected to a Digitimer constant current stimulator (DS7ASP; Digitimer, Welwyn Garden City, United Kingdom). The SCR
recording electrodes were attached to participants’ index andmiddle fingers on their non-dominant hand. The electric shockelectrodes were attached to the forearm of each participant’s

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in total the participants received the same concentration of theodorants as they had experienced during the rating phase. Oneof these cotton pads was inserted into the left- and the other into
the right-sided pocket of the facial mask, which the participant
subsequently wore.
During the Acquisition phase, all participants in the 2MTgroup were presented contextually with the predator odor
2MT (Kobayakawa and Kobayakawa, 2011). Each participant
in the Control group was presented contextually with the odor
that she had rated most similarly to 2MT. For both groups,
odors presented during the Acquisition phase shall be referred
to as “acquisition odors.” Overall for the Control group, thefollowing acquisition odors were used: konjac brine for six
participants, SeiroganTM for five participants, black bean paste
for three participants, Japanese pickled ginger brine for threeparticipants, and Jalapeño brine for one participant. Odors
were also presented contextually during the Extinction phaseof this paradigm (“extinction odors”). For each participant,this was the odor that she had rated most similarly to her
acquisition odor. The exception was that, despite being rated
similarly to their acquisition odor, 2MT was never used as theextinction odor for the control group. Overall, the following
extinction odors were used: Japanese pickled ginger brine for
12 participants, SeiroganTM for seven participants, Jalapeño
brine for six participants, konjac brine for five participants,
black bean paste for five participants, and roibos tea for one
participant. Each participant’s acquisition odor was presented
contextually during one session of the Test phase, and her
extinction odor was presented contextually in the other. Thisorder was counterbalanced between participants.
Participants’ ratings to their acquisition and extinction odors
are shown in Supplementary Figure S1. For each type ofrating (familiarity, pleasantness, and liking), a Group (2MT orControl) by Odor (their acquisition or extinction odor) repeatedmeasures analysis of variance (ANOVA) was conducted. The
results showed that, overall, the acquisition odors were rated
as less pleasant [F(1, 34) = 8.22, p = 0.007], less familiar [F(1,34) = 16.56, p < 0.001], and less liked [F(1, 34) = 9.42, p = 0.004]
than the extinction odors. These effects of Odor did not interact
with Group for pleasantness [F(1, 34) = 0.26, p = 0.616] orfor liking [F(1, 34) = 0.03, p = 0.866]. An interaction between
Odor and Group was found for familiarity [F(1, 34) = 4.31,
p = 0.046]. Importantly, however, follow-up independent-sample
t-tests showed no significant between-group differences forfamiliarity ratings of the acquisition odors [t(34) = −1.61,
p = 0.117] or for familiarity ratings of the extinction odors[t(34) = −0.11, p = 0.915]. Instead, and of no major consequencefor our experimental results, follow-up paired-sample t-tests
showed that the 2MT [t(17) = −3.95, p = 0.001], but not theControl group [t(17) = −1.59, p = 0.131], rated their acquisition
odor as less familiar than their extinction odor.

dominant hand. Participants completed all phases of theexperiment with their head in a headrest, with the aimof reducing their body movement and therefore movementrelated SCR artifacts.

Conditioned and Unconditioned Stimuli
We selected three male angry face photos from the ATR Facial
Expression Database (DB99; ATR-Promotions, Kyoto, Japan)1 to
be used as CS. We selected these stimuli because it has beenpreviously shown that fear responses are readily acquired to angry
face stimuli (Esteves et al., 1994) and that anger is more easily
perceived in male than in female faces (Becker et al., 2007).
For each participant, one of these photos was delegated as her“Main CS+” (for which she did not undergo extinction training),
a second as her “Extinction CS+” (for which she did undergo
extinction training), and the third as her “CS−.” These werecounterbalanced between participants. The US used was electric
shock, the strength of which was individually adjusted for eachparticipant to a level that she reported as uncomfortable but not
painful. For each instance of US presentation, the participants
experienced a series of five shocks over 50 ms. The average output
current was 32.50 mA (SD = 8.30 mA) from a source voltage of
200 V. The pulse width range was 50 µs. There was no differencein output current between the two groups of participants in either
the Acquisition or the Test phases (ps > 0.05).

Odors
In order to determine which odors to use for each individual,
prior to experimentation, we had each participant rate sevenodors for familiarity, pleasantness, and how much she liked themon a scale from −5 to + 5. Although “pleasantness” and “like”
may appear close to equivalent in English, these ratings werecompleted in Japanese, where the question for “pleasantness”
conveyed a meaning akin to questioning how objectively pleasantthe participants thought the odors were and the question for“like” conveyed a meaning akin to questioning how muchthey subjectively liked the odors. The rationale for including
both of these was that we considered that some people might
consider themselves to like (/dislike) a given odor, even while
they think that in general most people would dislike (/like) it.
The odorants used to make these odors were: 2MT, Jalapeño
brine, konjac brine, Japanese pickled ginger brine, black beanpaste, rooibos tea, and SeiroganTM (a Japanese herbal medicine).
For all odorants other than 2MT, 1.00 µl was applied to a
cotton pad that was presented in a jar for rating. For 2MT, asmaller amount was advised by its designer (R. Kobayakawa,
personal communication, April 6, 2016), and so 0.10 µl wasapplied to a cotton pad that was presented in a jar for rating.
Using this smaller amount of 2MT allowed for subjective ratings
to this which were comparable to those given to the otherodorants (see below). Throughout experimentation, the odors
were “contextually” presented in facial masks. Specifically, foreach participant for each “odor context,” the relevant odorant
was applied to two cotton pads. Half the amount presented fromthe odor rating phase was applied to each cotton pad so that1

Experimental Procedure
Acquisition Phase (Day 1)
The Acquisition phase consisted of two sessions. During eachsession, both of the CS+ s (Main CS+ and Extinction CS+)

http://www.atr-p.com/face-db.html

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were presented in nine trials each accompanied by the US andin another four trials each without the US. This intermittent
CS+ -US schedule was selected, in line with previous human fearconditioning studies (e.g., Norrholm et al., 2006; Schiller et al.,2008), with the hope that it would help to prevent participantsfrom habituating to the US (as was suggested by Sehlmeyer et al.,2009). The CS− was presented in nine trials without the US.
On 10 out of the total 35 trials per session, participants were
prompted to rate their expectation of receiving electric shock.
Specifically, this rating was prompted on two trials for eachCS+ with US, for two trials for each CS+ without US, and on
two trials for the CS− (see Figure 1A).
Within each trial (Figure 1B), a screen with a central fixation
point was presented for 12 s followed by a screen displaying
a CS for 4 s. On US trials, the US was presented during
the last 0.05 s of the CS presentation (Figure 1C). On thetrials where participants were prompted to rate US expectancy,
the following question was presented for 2 s in between the
fixation and the CS: “Do you think you will get an electric
shock on the next trial? Please reply as soon as possible after
the face image is presented.” Then, once the CS appeared onscreen, participants responded whether they thought they would“definitely get a shock,” “probably get a shock,” “probably not
get a shock,” or “definitely not get a shock” with their dominant
hand using the computer keyboard. Response key assignments
were counterbalanced between participants. The trial order was
randomized within each session. Each participant completed theAcquisition phase wearing a facial mask containing cotton pads
sprayed with her acquisition odor. Note that, even if unpleasant,these odors should not act, themselves, as US to be specificallyassociated with the CS+ s. This is because each participant’s “odormask” was worn throughout her entire Acquisition phase andtherefore it is unlikely that she would have learned to associate
the odor any more with the CS+ s than with the CS−. SCR data
were recorded throughout.

FIGURE 1 | (A) Experimental design of the main experiment. On Day 1(Acquisition phase), in her Acquisition Odor Context [2-methyl-2-thiazoline(2MT) or control odor], each participant learned to associate both conditioned
stimuli (CS)+ s, but not the CS−, with the unconditioned stimuli (US). Next, on
Days 2–4 (Extinction phase), in her Extinction Odor Context, each participant
was exposed to multiple presentations of the Extinction CS+ and the CS−,
with no US presentation. Finally, on Day 5, in the two sessions of the Test
phase, each participant was presented with all the CS. Reinstatement with the
US was completed at the beginning of each Test session. One of thesesessions was completed in the participant’s Acquisition Odor Context and theother in her Extinction Odor Context. This order was counterbalanced
between participants. (B) On each trial of the Acquisition and Test phases,
participants saw a central fixation point for 12 s. This was followed in somecases by a screen that appeared for 2 s with instructions for participants torate electric shock (US) expectancy once the CS appeared on screen. Finally,a CS was presented for 4 s. (C) On most CS+ trials during the Acquisition
phase, the US was presented during the last 0.05 s of CS+ presentation.

Extinction Phase (Days 2–4)
Participants completed three extinction sessions per day for threeconsecutive days to see if any effects found were robust enough tosurvive extensive extinction training (Figure 1A). In each session,
they were presented with the Extinction CS+ and the CS− 15times each in a randomized order. The US was not presented on
any trials. Within each trial, participants saw a central fixation
point on screen for 7 s, followed by a CS for 6 s, and then a blankscreen for 7 s. Each participant completed the Extinction phasewearing a facial mask containing cotton pads sprayed with her
extinction odor. SCR was recorded throughout.

remained on for 10 more min, during which participants were
instructed to simply relax, after which the Test session started.
Within each of the two sessions of the Test phase, participants
saw each of the three CSs seven times (21 trials in total). On
two trials for each CS, participants were prompted to rate their
US expectancy in the same manner as in the Acquisition phase.
Trials were presented in a random order, except that the CS− wasalways presented on the first trial (to capture the orienting effect;
Bouton, 2004; Koizumi et al., 2016), and the Main and Extinction
CS+ s were always presented second and third in random order.

Reinstatement and Test Phase (Day 5)
Prior to the Test phase, the strength of electric shock was
individually readjusted for each participant to a level that she
considered to be uncomfortable but not painful. There were
two sessions of the Test phase (Figure 1A), each of which waspreceded by a reinstatement procedure (Bouton, 2004). During
reinstatement, over the course of 1 s, participants were given four
series of five unsignaled electric shocks (one series every 250 ms),
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Because SCR is often found to be most prominent in the first
few trials of testing, we kept the US expectancy rating prompt
out of these trials to prioritize SCR measurement. The sequenceof events within Test phase trials was identical to those within
Acquisition phase trials except that the US was never presented(Figure 1B). During the Test phase, each participant wore afacial mask containing cotton pads sprayed with her acquisition
odor in one session and her extinction odor in the other. Thisorder was counterbalanced between participants. The SCR wasrecorded throughout.
FIGURE 2 | Results of the Acquisition phase. Skin conductance responses(SCRs) (peak-trough) were z-scored, log-transformed, and averaged acrossparticipants in each group. Unconditioned stimuli (US) expectancy ratings(from -5 to 5) were averaged across participants in each group. Overall,participants in both the 2-methyl-2-thiazoline (2MT) and Control groups weresimilarly successful in fear acquisition. For both SCR and US expectancy,
mean responses to the conditioned stimuli (CS)- were subtracted from thoseto the CS+ s to display fear-specific effects. Error bars represent standard
error of the mean. (A) The SCRs of both groups, were significantly greater tothe two CS+ s than to the CS−. This provides evidence for successfulacquisition of CS−US associations. (B) Likewise, the US expectancy ratingsof both groups, were significantly greater to the two CS+ s than to the CS.

Statistical Analyses
Skin Conductance Responses
SCRs for each phase of this experiment (Acquisition, Extinction,
and Test) were analyzed with the Matlab analysis software
Ledalab V3.4.92 . The data from each participant were down
sampled to 10 Hz and manually artifact corrected for eachphase (Boucsein et al., 2012). Then, optimized continuous
deconvolution analysis (Benedek and Kaernbach, 2010) was
performed. This analysis involves deconvolution of skinconductance data by the general physiological SCR shape toseparately extract continuous tonic and phasic drivers of skinconductance (Benedek and Kaernbach, 2010). SCR data from thephasic driver were standardized using a z-score transformation.
The SCR amplitude for each trial for each participant wascalculated by subtracting the trough from the peak data point
within the 1–4 s time window after CS onset. The data werenormalized using a log transformation (Braithwaite et al.,2015). The processed data were averaged separately for eachparticipant for each CS.

SCRs (for each participant to each CS) were the dependent
factors in both models. The first model had fixed effects of Group(2MT or Control group), CS (Main CS+, Extinction CS+, orCS−), and of the interaction between Group and CS. The second
model had the same fixed effects and a random intercept ofsubject. Data from 3 of the 36 participants (2 from the 2MTgroup) was excluded from these models because of technicalissues with skin conductance electrode during the Acquisition
phase. Because the US expectancy ratings of these 3 participants
showed they could clearly discriminate between the CS+ s andthe CS− (Supplementary Information), and because there wereno technical issues with the skin conductance electrodes for theseparticipants during the Test phase (which was of most interest),
their data were included in all other analyses. A likelihood ratio
test showed that the second model, which included a random
intercept of subject, explained SCR significantly better [Akaike’s
Information Criterion (AIC) = −20.27] than did the first model[AIC = 32.15; χ2 (1) = 54.41, p < 0.001]. This, and all subsequent
likelihood ratio tests, were computed using the Statistics andMachine Learning ToolboxTM for Matlab.
An ANOVA for the fixed effects of the second model showeda significant main effect of CS [F(2, 93) = 3.57, p = 0.032] (thisand all other ANOVAs of LME models were calculated using
the Statistics and Machine Learning ToolboxTM for Matlab). No
significant effect of Group [F(1, 93) = 0.85, p = 0.358] and no
significant Group by CS interaction [F(2, 93) = 0.28, p = 0.759]
were found, indicating that physiological responses did not differ
between groups in the Acquisition phase. Consistent with properacquisition of CS−US associations, a follow-up t-test showed that
SCRs were significantly greater to the Main CS+ [M = 0.67,
SD = 0.23; t(32) = 3.44, p = 0.002] and to the Extinction CS+[M = 0.67, SD = 0.30; t(32) = 3.06, p = 0.004] than they were to theCS− (M = 0.55, SD = 0.28). There was no significant difference in
SCRs to the two CS+ s [t(32) = 0.03, p = 0.973].

Unconditioned Stimulus Expectancy
Participants’ ratings that they thought they would “definitely geta shock,” “probably get a shock,” “probably not get a shock,” or“definitely not get a shock” were coded as ratings of 3, 2, 1, and0, respectively. The averages of these were then taken in eachrelevant phase to determine their US expectancy.

RESULTS OF THE MAIN EXPERIMENT
Skin Conductance Responses in the
Acquisition Phase
To ensure that participants learned to associate the CS+ (but not
the CS−) with the US, we examined the SCR in the Acquisition
phase that occurred in the 1–4 s window after each CS onset(Figure 2A). The SCR in this time window is expected to beuntainted by potential responses to the US, despite the US being
presented 3.95 s after the CS in some trials. This is because
the onset of the SCR is estimated to arrive at the sweat glandsof the fingers about 1.6 s (± 0.03) after electrical stimulation(Lim et al., 2003).
Two linear mixed-effects (LMEs) models were estimated andcompared to determine whether or not including a random
intercept of subject helped to better explain the data. The mean
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Unconditioned Stimulus Expectancy in
the Acquisition PhaseTo further ensure that participants had learned to associate theCS+ s (but not the CS−) with the US, we examined the ratings
that they gave in some trials of the Acquisition phase about how
likely they thought they were to receive the US. The same two
LME models that were used to analyze SCRs in the Acquisition
phase were run again, but with mean US expectancy ratings fromthe Acquisition phase (Figure 2B) as the dependent factor. There
was no significant difference between the models [χ2 (1) = 0,
p = 1.000; AIC = 210.91 for the first model, AIC = 212.91 forthe second model]. An ANOVA of the first model (which only
had the fixed effects) showed a significant main effect of CS [F(2,102) = 33.17, p < 0.001]. No significant main effect of Group[F(1, 102) = 0.02, p = 0.890] and no significant Group by CSinteraction [F(2, 102) = 0.10, p = 0.902] were found, indicatingthat US expectancy was equal for both groups of participants.
In evidence that participants had gained a proper
understanding of CS−US associations, follow-up t-tests showedthat mean US expectancy ratings were significantly greater to theMain CS+ [M = 2.29, SD = 0.65; t(35) = 7.58, p < 0.001] and theExtinction CS+ [M = 2.08, SD = 0.50; t(35) = 8.45, p < 0.001]
than they were to the CS− (M = 0.83, SD = 0.68). There was no
significant difference in mean US expectancy ratings between the
two CS+ s [t(35) = 1.80, p = 0.081], suggesting that the CS−US
associations were similarly learned for these.

FIGURE 3 | Results of the Test phase. Skin conductance responses (SCRs)(peak–trough) were z-scored, log-transformed, and averaged acrossparticipants in each group. Unconditioned stimuli (US) expectancy ratings(from -5 to 5) were averaged across participants in each group. Mean
responses to the conditioned stimuli (CS)- were subtracted from those to theCS+ s to display fear-specific effects. Furthermore, because no effects of theodor context used in the Test phase were found, mean responses wereaveraged across these for ease of display. Error bars represent standard errorof the mean. (A) Overall, for the 2-methyl-2-thiazoline (2MT) group, SCRs
were significantly stronger to the two CS+ s than to the CS−. No significant
differences were found for the Control group. For demonstrative purposes,
independent-sample t-tests were conducted to compare (CS+ – CS−)
between groups (collapsed across the two Odor Contexts used in the Test
phase). Both showed significant differences (**p < 0.01). (B) On average, bothgroups rated US expectancy as higher on the CS+ than on the CS− trials,
with no apparent difference between the groups.

Follow-up paired sample t-tests (collapsed for Odor Context)
were conducted using a Bonferroni adjusted alpha level of 0.008(0.05/6). The 2MT group were found to have significantly higher
SCRs to the Main CS+ [M = 0.83, SD = 0.47; t(17) = 5.14,
p < 0.001] and to the Extinction CS+ [M = 0.78, SD = 0.44;t(17) = 3.42, p = 0.003] than to the CS− (M = 0.58, SD = 0.32).
They showed no significant difference in SCRs to the two CS+[t(17) = 0.83, p = 0.421]. These results indicate that the 2MTgroup still displayed fear-like responses to the stimuli that had
been associated with electric shock. In contrast, the Control
group did not show significantly larger SCRs to the Main CS+[M = 0.75, SD = 0.56; t(17) = −0.87, p = 0.396] or the Extinction
CS+ [M = 0.71, SD = 0.58; t(17) = −1.53, p = 0.144] than they did
to the CS− (M = 0.82, SD = 0.67). They also showed no significant
difference in SCRs to the two CS+ s [t(17) = 0.66, p = 0.517].

Skin Conductance Responses in the Test
Phase
We examined SCRs from the Test phase that occurred four days
after the Acquisition phase to see if participants still had
greater SCRs to the CS+ s than to the CS− (Figure 3A
and Supplementary Figure S2). Each participant completed
two sessions of the Test phase: one in her Acquisition Odor
Context and one in her Extinction Odor Context (the order wascounterbalanced between participants).
We ran two LME models with mean SCRs from the Test
phase as the dependent factor. The first model had fixed effectsof Group (2MT or Control group), CS (Main CS+, Extinction
CS+, or CS−), Odor Context (Acquisition or Extinction Odor
Context), and of the interactions between Group, CS, and Odor
Context. The second model had the same fixed effects and arandom intercept of subject. Via likelihood ratio testing, thesecond model (AIC = 194.55) was shown to better explain mean
SCRs in the Test phase than the first model [χ2 (1) = 185.82,
p < 0.001; AIC = 378.37].
An ANOVA of the fixed effects of the second model showeda significant main effect of CS [F(2, 204) = 5.09, p = 0.007].
This was qualified by a significant CS by Group interaction [F(2,204) = 3.31, p = 0.038]. There was neither a significant main effectof Odor Context [F(1, 204) = 0.16, p = 0.692] nor any significant
interactions involving Odor Context [for its interaction with CS:
F(2, 204) = 0.37, p = 0.691; for its interaction with Group: F(1,204) = 0.29, p = 0.590; for the three-way interaction between Odor
Context, CS, and Group: F(2, 204) = 0.22, p = 0.803].

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Unconditioned Stimulus Expectancy in
the Test Phase
We examined whether participants still showed greater US
expectancy to the CS+ s than to the CS− in the Test phase(Figure 3B). We ran the same two LME models that were used toanalyze SCRs from the Test phase, but with mean US expectancy
ratings from the Test phase as the dependent factor. Likelihood
ratio testing showed the second model (which had the fixed
effects and a random intercept of subject; AIC = 514.19) tobetter explain US expectancy ratings from the Test phase than
the first model [which only had the fixed effects, χ2 (1) = 90.59,
p < 0.05; AIC = 602.78]. An ANOVA of the fixed effectsof the second model showed a significant main effect of CS[F(2, 203) = 3.50, p < 0.05]. No main effects of Group [F(1,6April 2020 | Volume 14 | Article 255

Taylor et al.

Threat Odor Strengthens Human Memory203) = 0.20, p = 0.653] or Odor Context [F(1, 203) = 0.00,
p = 1.000] were found to be significant. Likewise, no significant
interactions were found (for the interaction between CS andContext [F(2, 203) = 0.05, p = 0.949]; for the interaction between
CS and Group [F(2, 203) = 0.06, p = 0.945]; for the interaction
between Context and Group [F(1, 203) = 0.43, p = 0.514];
for the three-way interaction between Context, CS, and Group[F(2, 203) = 0.11, p = 0.896]).
Because the ANOVA revealed a significant main effect ofCS, we conducted follow-up paired sample t-tests (collapsed forGroup and Odor Context). A Bonferroni adjusted alpha levelof 0.008 (0.05/6) was implemented. Overall, participants showedsignificantly higher US expectancy ratings to the Main CS+[M = 1.11, SD = 0.83; t(35) = 3.06, p = 0.004] than to the CS−(M = 0.66, SD = 0.92). They also showed higher US expectancy
ratings to the Extinction CS+ (M = 1.03, SD = 0.86) than to
the CS− [t(35) = 2.33, p = 0.025], although this did meet theadjusted alpha level of significance. They showed no difference in
US expectancy ratings to the two CS+ s [t(35) = 1.06, p = 0.295].
These results indicate that both groups of participants explicitly
recalled which stimuli had been associated with shock. However,it must be noted that US expectancy ratings were few and weredeliberately collected only after the first few trials so as not
to interfere with SCR measurement (see section Experimental
Procedure). Therefore, we cannot rule out the possibility thatbetween-group or between-context differences occurred early onin each session.

teeth for 1 h before coming in. Participants were randomly
assigned to the 2MT context (2MT group) or the control odor
context (Control group). The data of two participants were
excluded from the analyses. The data of one participant were
excluded because she had extremely high baseline saliva levels
that were above the typical range for her age-group (Aardal
and Holm, 1995). The data of the other were excluded because,
unlike all other participants, she rated all the odors extremely
positively. Of the remaining 20 participants, 10 were in the
2MT group and 10 were in the Control group. There was no
significant difference in age between groups [for the 2MT group:M = 24.8, SD = 4.7; for the Control group: M = 24.2, SD = 4.9;t(18) = −0.28, p = 0.781].

Salivary Cortisol Collection and Analysis
Participants provided each saliva sample via the Salimetrics
passive drool method (Salimetrics LLC, State College, PA,
United States) with the aim of providing at least 1.5 ml foreach sample. Samples were stored at −80◦ C, then thawed
and centrifuged at 1,500 × g for 15 min at 4◦ C to remove
mucins and other particulate matter. Clear saliva samples
were aliquoted into new sample tubes and stored at −80◦ C
until the day of a cortisol assay. The saliva samples wereassayed for cortisol using an enzyme-linked immunosorbent
assay and the four-parameter non-linear regression standard
curve method recommended by the manufacturer of this assay(Salimetrics LLC, State College, PA, United States). Samples
were run in duplicate to ensure reliability. The cortisol data
for each participant were standardized, using a square roottransformation (Kobayashi and Miyazaki, 2015), before being put
into LMEs models.

MATERIALS AND METHODS FOR THE
FOLLOW-UP CORTISOL EXPERIMENT
Fear memory formation is enhanced by high levels ofcorticosteroids during learning (Roozendaal and McGaugh,2011). We therefore speculated that if a 2MT context causes
higher levels of corticosteroids in humans, as it has previously
been shown to do in mice (Isosaka et al., 2015), then thismight explain our above finding of stronger fear memory
formation in a 2MT learning context. We conducted a simple
follow-up experiment to examine this. Where not else specified,methods and materials were identical to those used in themain experiment.

Experimental Procedure
Each participant first came in and, after signing a consent
form, completed the same odor rating task as was completed
in the main experiment. The odor for each participant in
the Control group was then selected as that which she rated
most similarly to 2MT. These were black bean paste for threeparticipants, SeiroganTM for two participants, konjac brine
for two participants, Japanese pickled ginger brine for twoparticipants, and roibos tea for one participant. The odor for
all participants in the 2MT group was 2MT. Next, after theparticipant had completed the questionnaire mentioned above(which also asked for the exact time she woke up and when her
last menstrual cycle begun), the SCR electrodes were attached
to her. With her head in a headrest in order to reduce motion,
the participant then rested while looking at a blank screen for
15 min. After this, her baseline level of salivary cortisol wasmeasured. Subsequently, a facial mask containing an odorant(2MT or control odorant) was put on her, and her level ofsalivary cortisol was measured 10, 20, and 30 min subsequent
to this. All participants kept the facial masks on throughout
the experiment except when giving saliva samples. They simply
rested in the headrest and looked at a blank screen in between
saliva sample collections.

ParticipantsParticipants were 22 Japanese females with normal or corrected
to normal vision. Each participant was paid U6,000. To ensure
validity of cortisol measurements, only participants who took no
regular medication were recruited. Furthermore, all participants
were informed before coming in of a list of requirements.
A questionnaire before the start of the experiment ensured thatall participants had abided by these. The requirements wereas follows: Participants should not drink alcohol or take any
medication for 24 h before coming in; they should not smokeor drink anything with caffeine on the day of experimentation;
they should wake up before 9 a.m. on the day of experimentation;
they should not eat anything, drink anything but water, or
exercise for 2 h before coming in; they should not brush their

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Threat Odor Strengthens Human Memory

Baseline cortisol subgroups), a follow-up t-test examining thiseffect did not prove significant. The Bayes Factor for this t-test
was 0.8033 indicating that this (small) data set is not sufficient tosupport the null or the alternative hypothesis. Nonetheless, theinteraction between Group and Baseline Level of cortisol appearsof importance because when compared to a model which only
had the independent factor of Group (and a random intercept;
AIC = −200.76), likelihood ratio testing showed the model that
included the interaction (AIC = -229.54) to better explain the data[χ2 (1) = 30.78, p < 0.001].

RESULTS OF THE FOLLOW-UP
CORTISOL EXPERIMENTQuestionnaire and Odor Rating Results
Results of the questionnaire showed that factors which could
potentially have contributed to salivary cortisol levels did not
significantly differ between groups. Specifically, the amount ofhours since waking up (for the Control group, M = 6.39,
SD = 2.14; for the 2MT group, M = 6.33, SD = 1.46) and theamount of days since the beginning of their last menstrual cycle(for the Control group, M = 16.10, SD = 8.41; for the 2MTgroup, M = 15.60, SD = 6.95) were well-matched between groups.
Odor ratings were also similar between groups (Supplementary
Figure S3). Therefore, these factors are unlikely to have caused
any between-group differences in cortisol level.

DISCUSSION
Our results show that the predator odor 2MT enhances thepersistence of physiological aspects of human fear memory
associations learned within its context. Participants in this study
who learned to associate visual stimuli with electric shock in a
2MT context (2MT group) had greater fear-like SCRs to thesevisual stimuli in a later Test phase than did participants wholearned these associations in a control odor context (Control
group). This effect of 2MT appears to be robust because it was
found for the 2MT group in both odor contexts of the Test
phase, even to a CS+ which had undergone extinction training.
The preliminary findings of a follow-up experiment hint that
increased levels of cortisol in a 2MT context may provide a
potential biological basis for this effect.
Several results indicated that physiological aspects of fear
memories formed in the 2MT context were robust. First, fearlike SCRs learned in a 2MT context were strong enough not
only to simply last for 3 days (as evidenced in responses tothe Main CS+ during the Test phase; Figure 3A) but also
to resist 3 days of extensive extinction training (as evidenced
in responses to the Extinction CS+ during the Test phase;
Figure 3A). Second, the 2MT group displayed fear-like SCRs to
the CS+ s even in the Extinction Odor Context of the Test phase(Supplementary Figure S2), further indicating the persistenceof their fear responses. This is because, unlike acquisition
contexts which are thought to promote retrieval of CS−US
associations, extinction contexts are thought to promote retrievalof CS−no US associations (Bouton, 2004). Third, although thepreliminary results of a follow-up Long-Term Test (with identicalmethodology to the main Test; see Supplementary Information)
showed that the 2MT group’s SCRs and US expectancy ratings
were not significantly different from those of the Control group
3 months later, there was a numeric trend showing them to behigher (Supplementary Figure S4). Overall, the results indicatethat the mere presence of a 2MT context during learning
strengthened the formation of fear associations.
The corticosteroid levels of mice are increased upon exposure
to 2MT (Isosaka et al., 2015), and we therefore hypothesized thatthe same might be the case for humans. Cortisol is a hormone thatis released by the hypothalamus–pituitary–adrenal axis (HPAA)
under conditions of high stress/fear; it therefore serves as an
endocrine biomarker (Foley and Kirschbaum, 2010). Comparedto other biomarkers of stress/fear, cortisol has been particularly
linked to memory consolidation and is thus of particular interest

Cortisol Results
Cortisol data at each Time Point (10, 20, or 30 min after theapplication of the odor mask), as well as Baseline Level of salivary
cortisol (taken 15 min before application of odor masks), werestandardized using a square root transformation (Kobayashi and
Miyazaki, 2015). Then, two LME models were conducted toanalyze the salivary cortisol levels of participants subsequent
to application of their odor masks. The dependent variablein each model was standardized cortisol level (Figure 4). The
explanatory fixed factors of interest in both models were Group(2MT or Control group), Time Point, and their interaction. As an
additional fixed factor in both models, the interaction between
standardized Baseline Level of salivary cortisol and Group was
also included. This is because despite between-group differences
not being significant, Baseline Level of salivary cortisol appeared
to differ somewhat between groups, which is of potential concern
for such a small sample size. We therefore wished to account forvariance potentially explained by between-group differences inthis. The first model that we constructed only included these fixedfactors. The second included these fixed factors and a random
intercept of subject.
Likelihood ratio testing showed the second model (AIC = 229.54) to better explain cortisol levels than the first model[χ2 (1) = 27.43, p < 0.001; AIC = −204.11]. An ANOVA ofthe fixed effects of this second model showed a significant main
effect of Group [F(1, 55) = 54.54, p < 0.001]. This was qualified
by a significant interaction between Group and Baseline Levelof salivary cortisol [F(1, 55) = 73.19, p < 0.001]. No othersignificant main effects or interactions were found (ps > 0.05),
indicating that Time Point and the interaction between Time
Point and Group did not significantly contribute to the cortisol
levels. As can be seen in Figure 4, where participants in bothgroups have been median split into low and high Baseline cortisol
subgroups, there were no apparent between-group differences forparticipants with high Baseline Levels of cortisol (this may be due
to a ceiling effect). However, for the participants with low Baseline
Levels of cortisol, cortisol levels subsequent to induction of theodor context appear to be higher for participants in the 2MT thanin the Control group. Not surprisingly, given the small sample
size for this effect (five participants each for the 2MT/Control low

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Threat Odor Strengthens Human Memory

FIGURE 4 | Results of the follow-up cortisol experiment. Error bars represent standard error of the mean. Participants were median split into (A) those with lower,and (B) those with higher baseline levels of salivary cortisol. For participants with lower baseline levels, post- odor mask application salivary cortisol levels appear
higher for those in the 2MT than in the Control group. For participants with higher baseline levels, there were no obvious between-group differences, possibly due toa ceiling effect.

(Kobayakawa and Kobayakawa, 2011), and it has previously been
proposed that the sulfur metabolites of digested meat indicate
the general presence of a potentially dangerous carnivore nearby(Apfelbach et al., 2005). In evidence for this, mice have beenshown to make more fear-like responses around the odor of cats
who have been fed a carnivorous compared to a vegetarian diet
(Berton et al., 1998), and some animals have even been shown todisplay fear-like responses to the odor of a predator to which their
species was previously naive (Mella et al., 2016). Importantly,
even humans have been shown to have relatively high sensitivity
in detecting sulfur-based predator odors (Sarrafchi et al., 2013).
Therefore, although it has previously been speculated that odors
gain their affective value for humans via learned experience(Herz and Engen, 2010; Li, 2014), our findings suggest that someodors such as 2MT may instead be innately threat-relevant tohumans. They raise the questions: in what other scenarios, and towhat further extent, might background odors innately modulate
human affective and cognitive processes? what other odors might
induce similar effects?
In this study, we found that fear-like responses in the Test
phase differed depended on whether participants had completed
learning in the 2MT context but not dependent on whether(for the 2MT group) they were tested in the 2MT context. This
indicates that 2MT context enhanced the formation, rather than
the recall, of fear memories. In the real world, fear memories
are often formed in environments that are rich in backgroundolfactory information. Similar to our finding, threat-relevant
background odors in the real world may modulate the strengthof fear memories formed within their context. One example isthat flashbacks in people with post-traumatic stress disorders(PTSDs) are often reported to be triggered by threat-relevant
odors that were present in the initial context of a traumatic
experience (Vermetten and Bremner, 2003; Hinton et al., 2004).
It is possible that, rather than just triggering recall of thesememories, these threat-relevant odors may have contributed tothe overall strength of memory formation in the first place. Thus,

for this experiment. Therefore, to investigate whether 2MT affects
human levels of cortisol, in a follow-up experiment, we put
human participants in either a 2MT or a control odor context
and measured their salivary levels of cortisol at several subsequent
time points. An interaction between Group (2MT/Control) andbaseline levels of cortisol was found to significantly predict
salivary cortisol levels after participants were put into their odor
contexts. Possibly due to a ceiling effect, this appeared no higher
in a 2MT than in a control odor context for participants with highbaseline levels. However, for participants with low baseline levels,
salivary cortisol appeared higher subsequent to entering a 2MT
compared to a control odor context.
Higher cortisol levels in a 2MT context may at least partly
explain our results from the main experiment. Previous studies
have shown that if the corticosteroid levels of humans and nonhuman animals are increased during or soon after learning,then this leads to enhanced persistence of memories (Sandi andRose, 1994; Buchanan and Lovallo, 2001). Furthermore, if thiscorticosteroid increase is blocked, then this effect is prevented(Roozendaal and McGaugh, 1997). The mechanism behind thiseffect is thought to be, at least in part (for a detailed description,
see Roozendaal and McGaugh, 2011), that corticosteroids bindto receptors in the basolateral amygdala and potentiate thenoradrenaline signaling cascade, which in turn enhances memory
consolidation processes in other parts of the brain such as the
hippocampus. Future studies may directly examine whether a
2MT context enhances fear memory acquisition by engaging
these neural mechanisms.
Our participants had probably never experienced 2MT before,
and they rated it as unfamiliar, therefore it is unlikely that they
had preexisting conditioned threat-relevant associations with thisodor. Instead, our results may have arisen because 2MT’s context
is one within which humans are “innately prepared” to better
learn fear associations. This idea might seem strange, consideringthat 2MT was derived from the odor of an animal (fox) that does
not directly prey upon humans. However, 2MT is sulfur-based

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a better understanding of how odor contexts may modulate thestrength of fear memory formation may help to elucidate why fear
memories are formed so robustly in clinical cases such as PTSDs.
Because the use of a 2MT context during conditioning was
found to strengthen physiological aspects of fear memories, thisstudy provides a potential methodological advantage for future
researchers. Investigations in human studies are often precluded
because the ethically approvable intensity of US (e.g., shock)only leads to weakly formed fear memories. A 2MT context
that is subjectively rated to be equally aversive to some othercommon odors could therefore be utilized in future studies tohelp strengthen memories of CS−US associations.
It must be noted that in the Test phase of the main experiment,
while there was a significant between-group difference in
SCRs, there was no significant between-group difference in
US expectancy ratings. On average, all participants had higher
US expectancy ratings to the CS+ s than to the CS− Thisfinding is of potential interest because it fits with the growing
literature, suggesting that physiological and self-reported fear
memory responses do not always align (Knight et al., 2003)
and sometimes can even be dissociated (Bechara et al., 1995).
It has been proposed that physical responses to threat might
be controlled by a different neural circuit to that which
controls the conscious feeling of fear (LeDoux and Pine,
2016; Taschereau-Dumouchel et al., 2019); Our current results
may demonstrate the effect of 2MT context specifically on
the neural circuit controlling physiological fear memories.
However, because of the experimental design used, we cannot
rule out the possibility that between-group differences in
US expectancy occurred right at the very beginning of the
Test phase sessions.
Overall, this study showed that human fear-like responses
to visual stimuli that were associated with threat were more
persistent when learned in a predator odor context. This may
be indicative of “innate preparedness” for fear learning incontexts reminiscent of threats from our evolutionary past.
The results of a preliminary follow-up test suggest that thismay occur because the predator odor context raises humancortisol levels, which, during learning, is known to lead tostronger memory consolidation. These results indicate thatthe human olfactory system might have more threat-relevant
evolutionary functions that might impact more upon our daily
lives than was previously considered. Future experimenters
might wish to consider utilizing 2MT contexts as a way tostrengthen participants’ CS−US associations. Taken together, ourresults accord with amassing warnings (Sarafoleanu et al., 2009;
Stevenson, 2010) that the human sense of olfaction should not
be underestimated.

DATA AVAILABILITY STATEMENTREFERENCES

Bechara, A., Tranel, D., Damasio, H., Adolphs, R., Rockland, C., and Damasio,
A. R. (1995). Double dissociation of conditioning and declarative knowledge
relative to the amygdala and hippocampus in humans. Science 269, 1115–1118.
doi: 10.1126/science.7652558
Becker, D. V., Kenrick, D. T., Neuberg, S. L., Blackwell, K. C., andSmith, D. M. (2007). The confounded nature of angry men and happy
women. J. Pers. Soc. Psychol. 92, 179–190. doi: 10.1037/0022-3514.92.
2.179

The datasets generated for this study are available on request to
the corresponding author.

ETHICS STATEMENT
The studies involving human participants were reviewed andapproved by Ethics Committee of the Review Board of Advanced
Telecommunications Research Institute International, Japan.
The participants provided their written informed consent toparticipate in this study.

AUTHOR CONTRIBUTIONSAll of the authors contributed to the experimental design. Data
collection and analysis for the Main experiment was conductedby JT. Data collection and analysis for the follow-up cortisol
experiment was conducted by JT with much assistance fromAN and HS. The manuscript was written by JT and AK, in
consultation with and with much advice and input from all otherauthors. All authors reviewed the manuscript.

FUNDING
This work was supported by the ImPACT Program of Council
for Science, Technology and Innovation (Cabinet Office,
Government of Japan), by the Brain/Mind Beyond program of theJapan Agency for Medical Research and Development (AMED)(JP19dm0307008), by JT presto (JPMJPR18J3), and by Versus
Arthritis: 21537.

ACKNOWLEDGMENTS
We thank Kaori Nakamura for her help in scheduling andconducting the experiments, Nobuo Hiroe and Hiroki Moriya for
their assistance with equipment, and Wei-Chuan Chang for her
help in collecting and analyzing cortisol samples.

SUPPLEMENTARY MATERIALThe Supplementary Material for this article can be foundonline at: https://www.frontiersin.org/articles/10.3389/fnins.
2020.00255/full#supplementary-material

Aardal, E., and Holm, A. (1995). Cortisol in Saliva — Reference ranges and relation
to cortisol in serum. Eur. J. Clin. Chem. Clin. Biochem. 33, 927–932.
Apfelbach, R., Blanchard, C. D., Blanchard, R. J., Hayes, R. A., and McGregor,
I. S. (2005). The effects of predator odors in mammalian prey species: a review
of field and laboratory studies. Neurosci. Biobehav. Rev. 29, 1123–1144. doi:
10.1016/j.neubiorev.2005.05.005

Frontiers in Neuroscience | www.frontiersin.org

10

April 2020 | Volume 14 | Article 255

Taylor et al.

Threat Odor Strengthens Human Memory

hierarchy between innate and learned fear. Cell 163, 1153–1164. doi: 10.1016/j.
cell.2015.10.047
Jones, S. V., Heldt, S. A., Davis, M., and Ressler, K. J. (2005). Olfactory-mediatedfear conditioning in mice: simultaneous measurements of fear-potentiatedstartle and freezing. Behav. Neurosci. 119, 329–335. doi: 10.1037/0735-7044.119.
1.329
Kass, M. D., Rosenthal, M. C., Pottackal, J., and McGann, J. P. (2013). Fear learningenhances neural responses to threat-predictive sensory stimuli. Science 342,
1389–1392. doi: 10.1126/science.1244916
Kirk-Smith, M., van Toller, C., and Dodd, G. (1983). Unconscious odour
conditioning in human subjects. Biol. Psychol. 17, 221–231. doi: 10.1016/03010511(83)90020-0
Knight, D. C., Nguyen, H. T., and Bandettini, P. A. (2003). Expression of
conditional fear with and without awareness. Proc. Natl. Acad. Sci. U.S.A. 100,
15280–15283. doi: 10.1073/pnas.2535780100
Kobayakawa, K., and Kobayakawa, R. (2011). Animal Repellents. U.S. Patent WO
2011096575A1.
Kobayakawa, K., Kobayakawa, R., Matsumoto, H., Oka, Y., Imai, T., and Ikawa,M. (2007). Innate versus learned odour processing in the mouse olfactory bulb.
Nature 450, 503–508. doi: 10.1038/nature06281
Kobayashi, H., and Miyazaki, Y. (2015). Distribution characteristics of salivary
cortisol measurements in a healthy young male population. J. Physiol.
Anthropol. 34:30. doi: 10.1186/s40101-015-0068-0
Koizumi, A., Amano, K., Cortese, A., Shibata, K., Yoshida, W., Seymour, B., et al.
(2016). Fear extinction without fear: Direct reinforcement of neural activity
bypasses the need for conscious exposure. Nat. Hum. Behav. 1, 1–7. doi: 10.
1038/s41562-016-0006
LeDoux, J. E. (2019). The Deep History of Ourselves: The Four-Billion-Year Story of
How We Got Conscious Brains. New York, NY: Viking Press.
LeDoux, J. E., and Pine, D. S. (2016). Using neuroscience to help understandfear and anxiety: a two-system framework. Am. J. Psychiatry 173, 1083–1093.
doi: 10.1176/appi.ajp.2016.16030353
Li, W. (2014). Learning to smell danger: acquired associative representation of
threat in the olfactory cortex. Front. Behav. Neurosci. 8:98. doi: 10.3389/fnbeh.
2014.00098
Lim, C., Seto-Poon, M., Clouston, P., and Morris, J. (2003). Sudomotor nerve
conduction velocity and central processing time of the skin conductance
response. Clin. Neurophysiol. 114, 2172–2180. doi: 10.1016/s1388-2457(03)00204-9
Mella, V. S. A., Cooper, C. E., and Davies, S. J. J. F. (2016). Effects of historicallyfamiliar and novel predator odours on the physiology of an introduced prey.
Curr. Zool. 62, 53–59. doi: 10.1093/cz/zov005
Norrholm, S. D., Jovanovic, T., Vervliet, B., Myers, K. M., Davis, M., Rothbaum,
B. O., et al. (2006). Conditioned fear extinction and reinstatement in a humanfear-potentiated startle paradigm. Learn Mem. 13, 681–685. doi: 10.1101/lm.
393906
Ohman, A., and Mineka, S. (2001). Fears, phobias, and preparedness: toward an
evolved module of fear and fear learning. Psychol. Rev. 108, 483–522. doi:
10.1037/0033-295x.108.3.483
Parma, V., Ferraro, S., Miller, S. S., Åhs, F., and Lundström, J. N. (2015).
Enhancement of odor sensitivity following repeated odor and visual fear
conditioning. Chem. Senses. 40, 497–506. doi: 10.1093/chemse/bjv033
Paschall, G. Y., and Davis, M. (2002). Olfactory-mediated fear-potentiated startle.
Behav. Neurosci. 116, 4–12. doi: 10.1037/0735-7044.116.1.4
Richardson, R., Vishney, A., and Lee, J. (1999). Conditioned odor potentiation ofstartle. Behav. Neurosci. 113, 787–794. doi: 10.1037/0735-7044.113.4.787
Roozendaal, B., and McGaugh, J. L. (1997). Glucocorticoid receptor agonist
and antagonist administration into the basolateral but not central amygdala
modulates memory storage. Neurobiol. Learn. Mem. 67, 176–179. doi: 10.1006/
nlme.1996.3765
Roozendaal, B., and McGaugh, J. L. (2011). Memory modulation. Behav. Neurosci.
125, 797–824. doi: 10.1037/a0026187
Sandi, C., and Rose, S. P. R. (1994). Corticosterone enhances long-term
potentiation in one-day-old chick trained in a weak passive avoidance learning
paradigm. Brain Res. 647, 106–112. doi: 10.1016/0006-8993(94)91404-4
Sarafoleanu, C., Mella, C., Georgescu, M., and Perederco, C. (2009). The
importance of the olfactory sense in the human behavior and evolution. J. Med.
Life 2, 196–198.

Benedek, M., and Kaernbach, C. (2010). A continuous measure of phasic
electrodermal activity. J. Neurosci. Methods 190, 80–91. doi: 10.1016/j.
jneumeth.2010.04.028
Berton, F., Vogel, E., and Belzung, C. (1998). Modulation of mice anxiety inresponse to cat odor as a consequence of predator’s diet. Physiol. Behav. 65,
247–254. doi: 10.1016/s0031-9384(98)00126-7
Blanchard, D. C., Markham, C., Yang, M., Hubbard, D., Madarang, E., andBlanchard, R. J. (2003). Failure to produce conditioning with low-dosetrimethylthiazoline or cat feces as unconditioned stimuli. Behav. Neurosci. 117,
360–368. doi: 10.1037/0735-7044.117.2.360
Blanchard, R. J., Yang, M., Li, C.-I., Gervacio, A., and Blanchard, C. (2001). Cue
and context conditioning of defensive behaviors to cat odor stimuli. Neurosci.
Biobehav. Rev. 25, 587–595. doi: 10.1016/S0149-7634(01)00043-4
Boucsein, W., Fowles, D. C., Grimnes, S., Ben-Shakhar, G., Roth, W. T.,
Dawson, M. E., et al. (2012). Publication recommendations for electrodermal
measurements. Psychophysiology 49, 1017–1034. doi: 10.1111/j.1469-8986.2012.
01384.x
Bouton, M. E. (2004). Context and behavioral processes in extinction. Learn. Mem.
11, 485–494. doi: 10.1101/lm.78804
Brainard, D. H. (1997). The psychophysics toolbox. Spat. Vis. 10, 433–436. doi:
10.1163/156856897x00357
Braithwaite, J. J., Watson, D. G., Jones, R., and Rowe, M. (2015). A Guide forAnalysing Electrodermal Activity (EDA) & Skin Conductance Responses (SCRs)
for Psychological Experiments. Available online at: https://www.birmingham.ac.
uk/Documents/college-les/psych/saal/guide-electrodermal-activity.pdf.
Buchanan, T. W., and Lovallo, W. R. (2001). Enhanced memory foremotional material following stress-level cortisol treatment in humans.
Psychoneuroendocrinology 26, 307–317. doi: 10.1016/s0306-4530(00)00058-5
Chen, D., Katdare, A., and Lucas, N. (2006). Chemosignals of fear enhancecognitive performance in humans. Chem. Senses 31, 415–423. doi: 10.1093/
chemse/bjj046
Chentsova-Dutton, Y. E., and Tsai, J. L. (2007). Gender differences in emotional
response among European Americans and Hmong Americans. Cogn. Emot. 21,
162–181. doi: 10.1080/02699930600911333
Dalla, C., and Shors, T. J. (2009). Sex differences in learning processes of classicaland operant conditioning. Physiol. Behav. 97, 229–238.
Eisdorfer, C., Doerr, H. O., and Follette, W. (1980). Electrodermal reactivity: ananalysis by age and sex. J. Human Stress 6, 39–42. doi: 10.1080/0097840X.1980.
9936107
Endres, T., and Fendt, M. (2007). Conditioned behavioral responses to a context
paired with the predator odor trimethylthiazoline. Behav. Neurosci. 121,
594–601. doi: 10.1037/0735-7044.121.3.594
Epple, G., and Herz, R. S. (1999). Ambient odors associated to failure influencecognitive performance in children. Dev. Psychobiol. 35, 103–107. doi: 10.1002/(sici)1098-2302(199909)35:2<103::aid-dev3>3.0.co;2-4
Esteves, F., Parra, C., Dimberg, U., and Ohman, A. (1994). Nonconscious
associative learning: pavlovian conditioning of skin conductance responses tomasked fear-relevant facial stimuli. Psychophysiology 31, 375–385. doi: 10.1111/
j.1469-8986.1994.tb02446.x
Foley, P., and Kirschbaum (2010). Human hypothalamus-pituitary-adrenal axis
responses to acute psychosocial stress in laboratory settings. Neurosci. Biobehav.
R. 35, 91–96. doi: 10.1016/j.neubiorev.2010.01.010
Hermann, C., Ziegler, S., Birbaumer, N., and Flor, H. (2002). Psychophysiologicaland subjective indicators of aversive pavlovian conditioning in generalizedsocial phobia. Biol. Psychiatry 52, 328–337. doi: 10.1016/s0006-3223(02)01385-9Herz, R. S., and Engen, T. (2010). Odor memory: review and analysis. Psychon. Bull.
Rev. 3, 300–313. doi: 10.3758/BF03210754
Hinton, D. E., Pich, V., Chhean, D., Pollack, M. H., and Barlow, D. H.
(2004). Olfactory-triggered panic attacks among Cambodian refugees attendinga psychiatric clinic. Gen. Hosp. Psychiatry 26, 390–397. doi: 10.1016/j.
genhosppsych.2004.04.007
Inslicht, S. S., Metzler, T. J., Garcia, N. M., Pineles, S. L., Milad, M. R., Orr,S. P., et al. (2013). Sex differences in fear conditioning in posttraumaticstress disorder. J. Psychiatr. Res. 47, 64–71. doi: 10.1016/j.jpsychires.2012.
08.027
Isosaka, T., Matsuo, T., Yamaguchi, T., Funabiki, K., Nakanishi, S., Kobayakawa,R., et al. (2015). Htr2a-Expressing cells in the central amygdala control the

Frontiers in Neuroscience | www.frontiersin.org

11

April 2020 | Volume 14 | Article 255

Taylor et al.

Threat Odor Strengthens Human Memory

correlates. Mol. Psychiatry doi: 10.1038/s41380-019-0520-3 [Epub ahead ofprint].
Vermetten, E., and Bremner, J. D. (2003). Olfaction as a traumatic reminder inposttraumatic stress disorder: case reports and review. J. Clin. Psychiatry 64,
202–207. doi: 10.4088/jcp.v64n0214
Wang, Y., Cao, L., Lee, C. Y., Matsuo, T., Wu, K., Asher, G., et al. (2018). Largescale forward genetics screening identifies Trpa1 as a chemosensor for predator
odor-evoked innate fear behaviors. Nat. Commun. 9:2041. doi: 10.1038/s41467018-04324-3
Wisman, A., and Shrira, I. (2015). The smell of death: evidence that putrescine
elicits threat management mechanisms. Front. Psychol. 6:1274. doi: 10.3389/
fpsyg.2015.01274

Sarrafchi, A., Odhammer, A. M. E., Hernandez Salazar, L. T., and Laska, M. (2013).
Olfactory sensitivity for six predator odorants in CD-1 mice, human subjects,
and spider monkeys. PLoS ONE 8:e80621. doi: 10.1371/journal.pone.0080621
Schiller, D., Levy, I., Niv, Y., LeDoux, J. E., and Phelps, E. A. (2008). From fear
to safety and back: reversal of fear in the brain. J. Neurosci. 28, 11517–11525.
doi: 10.1523/JNEUROSCI.2265-08.2008
Schneider, F., Weiss, U., Kessler, C., Müller-Gärtner, H. W., Posse, S., Salloum,J. B., et al. (1999). Subcortical correlates of differential classical conditioningof aversive emotional reactions in social phobia. Biol. Psychiatry 45, 863–871.
doi: 10.1016/S0006-3223(98)00269-8
Sehlmeyer, C., Schöning, S., Zwitserlood, P., Pfleiderer, B., Kircher, T., Arolt,
V., et al. (2009). Human fear conditioning and extinction in neuroimaging:a systematic review. PLoS ONE 4:e5865. doi: 10.1371/journal.pone.000
5865
Sevelinges, Y., Gervais, R., Messaoudi, B., Granjon, L., and Mouly, A. (2004).
Olfactory fear conditioning induces field potential potentiation in rat olfactorycortex and amygdala. Learn Mem. 11, 761–769. doi: 10.1101/lm.83604
Shionoya, K., Hegoburu, C., Brown, B. L., Sullivan, R. M., Doyère, V., Mouly, A. M.,
et al. (2013). It’s time to fear! Interval timing in odor fear conditioning in rats.
Front. Behav. Neurosci. 7:128. doi: 10.3389/fnbeh.2013.00128
Stevenson, R. J. (2010). An initial evaluation of the functions of human olfaction.
Chem. Senses. 35, 3–20. doi: 10.1093/chemse/bjp083
Taschereau-Dumouchel, V., Kawato, M., and Lau, H. (2019). Multivoxel patternanalysis reveals dissociations between subjective fear and its physiological

Frontiers in Neuroscience | www.frontiersin.org

Conflict of Interest: The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interest.
Copyright © 2020 Taylor, Lau, Seymour, Nakae, Sumioka, Kawato and Koizumi.
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