1. Supplementary background
Maternal odor attachment learning
In order for an infant rat pup to survive, it must learn to identify its mother’s dietdependent odor, so that it can approach and nipple attach to the mother. Natural maternal
odor was originally proposed to be a pheromone. However, decades of research have
shown that it is learned and odor specific (Galef and Kaner, 1980; Leon, 1992; Teicher
and Blass, 1977; Hofer et al, 1976; Pedersen et al, 1982; Moriceau et al, 2009; Sullivan et
al, 1990; Logan et al, 2012). Maternal odor learning begins in the womb, via learning of
the mother’s amniotic fluid. However, postnatal learning of maternal odor continues in
the nest, because the maternal odor is diet-dependent (Leon, 1975, 1992). Since the
maternal odor in rats is diet-dependent, pups will show a clear preference for the odor of
their mother and another mother equally, due to their identical diets. Published work from
our laboratory also demonstrates that pups show a very strong preference for natural
maternal odor, whether or not it is the odor of their own mother (Raineki et al, 2010).
If the natural maternal odor is eliminated through a special diet, so that pups never
experience the odor postnatally, pups fail to respond to the natural maternal odor as
assessed by behavioral and neural responses (Sullivan et al, 1990). Importantly, a novel
conditioned odor can reinstate behavioral and neural responses typically induced by
natural maternal odor, simply by placing the novel odor on the mother during motherinfant interactions (Sullivan et al, 1990; Teicher et al, 1978). Thus, the neonatal learning
system underlying maternal odor attachment learning is so robust, that learning can occur
through the simple pairing of an odor and sensory stimulation from the mother (Raineki
et al, 2010). Interestingly, a conditioned odor can acquire properties similar to maternal
odor, via controlled classical conditioning experiments performed outside the nest
without the mother (Johanson and Teicher, 1980; Moriceau et al, 2009; Sullivan et al,
2000a, 2000b). The pairing of a novel odor with warmth, nursing, or sensory stimulation
(i.e. stroking to mimic grooming) produces a learned odor preference, so that the odor
guides nipple attachment and mother-pup interactions (Brake, 1981; Galef and Sherry,
1973; Pedersen et al, 1982; Wilson and Sullivan, 1994). Interestingly, odor attachment
learning can occur in early infancy even when a novel odor is paired with painful stimuli.
This likely occurs to ensure pup attachment and survival, even if the caregiver is a source
of pain to the infant (Hofer and Sullivan, 2001; Roth and Sullivan, 2005).
The enduring value of maternal odor
Our laboratory has analyzed the role of odors learned during infant odor-shock or
odor-stroke conditioning in modulating infant and adult behavior. Following this
conditioning, pups display a strong odor preference for this conditioned odor, as the odor
induces approach responses in pups, and can control nipple attachment and social
interactions with the mother (Camp and Rudy, 1988, Raineki et al, 2010, Roth and
Sullivan, 2005; Sullivan et al, 1990, 2000b). Thus, the peppermint-conditioned odor
acquires comparable value to natural maternal odor. Furthermore, the conditioned odor
appears to retain value into adulthood, although the behaviors it controls change from
mother–infant interactions to behaviors important in adulthood. Specifically, the
conditioned odor has been shown to rescue later life deficits produced by early-life abuse,
such as depressive-like behaviors (forced swim test and sucrose preference test;
Sevelinges et al, 2007, 2011). In these previous manuscripts, we determined that the odor
must be paired with shock in order to produce an odor that is capable of controlling the
infants’ behavior towards the mother and also modulate depressive-like behavior in
adulthood. The peppermint odor that was used for the infant conditioning acquired
maternal odor qualities only for the animals that received paired odor-shock conditioning,
but not for unpaired odor-shock, or odor-only control animals. Importantly, the data also
indicated that all learning controls (odor-only, shock-only, unpaired) were not different
from naïve animals (no conditioning), suggesting that the factors associated with the
experimental manipulations, such as the maternal separation, did not affect that
parameters analyzed.
Nevertheless, we have demonstrated that receiving unpaired odor-shock
conditioning during infancy, which we believe is a model for unpredictable trauma in
infancy, increases anxiety-like behaviors in the adult (Sarro et al, 2014; Tyler et al, 2007).
Indeed, our data indicate that infant paired odor-shock is a model that can be used to
investigate how predictable early-life adversity may lead to depressive-like behaviors and
that unpaired odor-shock is a model that can be used to investigate how unpredictable
early-life adversity may lead to anxiety-like behaviors. However, the animals that
received unpaired odor-shock conditioning in infancy show no preference for the
peppermint odor in infancy. Likewise, the odor has no enduring effects on the unpaired
animals’ behavior and/or neural activity, as it has not acquired the value of the maternal
odor. The current manuscript demonstrates for the first time that the natural maternal
odor rescues depressive-like behaviors following early-life abuse, in the same manner as
odors conditioned in early infancy. Furthermore, we expand our findings in the
modulation of adult behaviors by early-life abusive attachment cues to social and sexual
behaviors.
Assessment of sexual motivation involved a behavioral task that our laboratory
has not investigated previously, and because receiving unpaired odor-shock conditioning
in infancy could affect the sexual performance in adulthood, we have added
supplementary results where we compare the group that received paired odor-shock
conditioning in infancy with the learning controls (unpaired and odor only; see below).
Animal models of abusive attachment
Our laboratory employs two rodent models of early-life abuse, which are used to
examine the infant response to abuse within the attachment system and the development
of later-life neurobehavioral deficits following abuse. The first model is a naturalistic
abuse paradigm where the mother handles her pups roughly when provided with
insufficient bedding for nest building (Hill et al, 2014; Ivy et al, 2008; Raineki et al,
2010, 2012; Roth and Sullivan, 2005). This impoverished environment results in frequent
attempts at nest building, trampling, and rough handling of pups, as well as decreased
nursing, however typical weight gain occurs (Raineki et al, 2010; Roth and Sullivan,
2005). The second model uses infant odor-shock conditioning to paradoxically produce
an odor that is preferred by infant rat pups (Camp and Rudy, 1988; Haroutunian and
Campbell, 1979; Roth and Sullivan, 2005). Importantly, this neutral odor paired with
shock in early infancy acquires the same value of natural maternal odor, and can control
mother-pup social behavior, despite the association of the odor with aversive shock
presentations. Furthermore, associative learning of odor-shock pairings before postnatal
day (PN) 10 uses the same neural pathway the infant rat naturally uses to learn maternal
odor (Landers and Sullivan, 2012; Moriceau and Sullivan 2006; Raineki et al., 2010).
Lastly, odor-shock conditioning provides a more controlled adverse environment when
modeling early-life abuse, and allows assessment of changes in the brain based
exclusively on aversive stimulation. The simultaneous use of our naturalistic (abusive
rearing) and experimentally controlled (odor-shock) models of early-life abuse provides
great insight into the mechanisms by which abuse produces enduring neurobehavioral
deficits, and how early-life attachment cues acquire their enduring value.
Local field potential recordings
Local field potentials (LFPs) represent a measure of summed or cooperative
synaptic activity within the region around a recording electrode. Synaptic activity within
a specific region often occurs in a cooperative pattern of oscillations that can be divided
into different frequency bands. These specific frequency bands – including theta, beta and
gamma – are believed to reflect both different underlying cellular mechanisms and circuit
functions depending on the region of interest (Buzsáki, 2006). Below is a brief
description of how the specific frequency bands within the LFP oscillation are often
referred, with a special focus on the higher frequency oscillations, gamma (35-90Hz), as
these are specifically altered in the odor-evoked response of the amygdala in animals
with early-life abuse.
Theta
Slow-wave activity or theta oscillations in adults are often associated with
endogenous mechanisms of sleep and homeostasis (Steriade et al, 1993; Tononi and
Cirelli, 2006), and specifically have been found in rodents during REM sleep as well as
during a transient sleep state characterized by synchronized whisker twitching
(Vanderwolf, 1969; Nicolelis et al, 1995; Fanselow and Nicolelis, 1999; Gervasoni et al,
2004). Slow-wave activity has also been demonstrated to be critically involved in
memory consolidation and synaptic homeostasis (Tononi 2009; Diekelmann and Born,
2010).
Beta
Alongside gamma oscillations, beta oscillations are found during wake states and
arousal (Steriade et al, 1993). Often these higher frequency oscillations are thought to be
associated with information transfer across brain regions (Buzsáki, 2006; Engel et al,
2001). These kinds of activity are widely observed in sensorimotor regions and related to
performing motor actions, such as exploratory behavior (Murthy and Fetz, 1992; Sanes
and Donoghue, 1993). Notably, beta oscillations within the olfactory bulb have been
associated with odor sampling in rats (Ravel et al, 2003).
Gamma
Most relevant to the present study, gamma frequency oscillations are commonly
associated with reverberatory activity in local excitatory-inhibitory circuits during wake
states, and are especially sensitive to GABAergic interneuron function in many brain
areas (Lasztóczi and Klausberger, 2014; Traub et al, 1996; Buzsáki, 2006; Cardin et al,
2009; Volman et al, 2011; Baldauf and Desimone, 2014) including the amygdala
(Sinfield and Collins, 2006). This is interesting since we show an importance of
GABAergic function in the mechanisms and consequences of early abusive learning
(Thompson et al, 2008). Additionally, gamma oscillations have been associated with
cognitive functions such as attention, integration of sensory and multisensory signals, and
memory formation (Engel et al, 2001; Jensen et al, 2007). In the amygdala, there are
enhanced gamma oscillations in response to learned stimuli (Headley and Weinberger,
2013), and evidence suggests that they may coordinate local amygdala neural activity
with activity in other cortico-limbic areas (Bauer et al, 2007).
Significance of finding a specific difference within the Gamma band
While we obtained and compared the full spectrum of oscillation frequencies
across the animal conditions, we focused our discussion and presentation of the data on
the higher frequency gamma frequency oscillations (35-90Hz) because this was where
the animal conditions differed. Thus, the odor-specific enhancement of amygdala gamma
oscillations to the odor learned during abusive experience in infancy may reflect longlasting changes in amygdala GABAergic function. In fact, previous work has
demonstrated changes in amygdala paired-pulse inhibition following early-life abusive
experience, which also implicates a change in GABAergic circuitry (Sevelinges et al,
2007, 2011; Rincón-Cortés et al, unpublished observations). Furthermore, amygdala
GABAergic function undergoes dramatic developmental changes during the period in
which the animals used in the present study were exposed to early-life abuse (Thompson
et al, 2008; Ehrlich et al, 2013), suggesting that these may have been particularly
vulnerable during the manipulation.
2. Supplementary materials and methods
Subjects
Male Long-Evans rats (Harlan Labs) born and bred in our colony were used in the
experiments. The animals were housed (polypropylene cages 34 x 29 x 17 cm, wood
shavings, ad libitum food and water) in a temperature (201°C) and light (6:00-18:00
hours) controlled room. The day of birth was considered PN0 and litters were culled to 12
pups (6 males, 6 females) on PN1. Procedures were approved by the Institutional Animal
Care and Use Committee, which follow National Institutes of Health guidelines.
Infant abuse paradigms
Naturalistic abusive mother paradigm. The mother and her pups were housed in
a cage with limited nesting/bedding material from PN8-12. Specifically, on the morning
of PN8 all pups and the mother were transferred to a clean cage with limited
nesting/bedding material that consisted of a 1.2 cm layer of wood shavings. The animals
remained in this limited bedding environment until the afternoon of PN12. During this
period, the maternal behavior was observed daily for 30 min. The behaviors observed
included the time that the mother spent in the nest and nursing (nipple attached, but not
necessarily feeding), the frequency of rough handling (i.e. mother aggressively grooming
pups, transporting pups by limb), stepping or jumping on the pups, and nest building.
Additionally, the frequency of the pups’ vocalizations was also recorded. Similar to our
previous data (Raineki et al, 2010, 2012), this limited bedding environment (Table 1)
decreased the mothers’ abilities to construct nests, which resulted in frequent attempts at
nest building (t(11)=3.91 p<0.003), more time spent away from the nest (t(11)=3.91
p<0.003), an increased frequency of stepping or jumping on the pups (t(11)=2.95 p<0.05),
and rough handling of pups (t(11)=2.04 p=0.06). Consequently, pups spent less time
nursing (t(11)=4.61 p<0.001) and had increased vocalizations (t(11)=2.61 p<0.03). Despite
the reduction in the time nipple attached, being reared by an abusive mother did not lead
to a reduction in pups’ body weight at PN12 (t(10)=0.01 p=0.99). We have not yet
assessed if animals reared by an abusive mother show a difference in feeding bouts;
however, since no reduction in body weight is found, it seems that the pups are not
malnourished.
Olfactory classical conditioning paradigm. Beginning at PN8, pups were odorshock conditioned daily for 5 consecutive days. Pups were removed from the mother,
who stayed in the home cage, and were transferred to a different room where they were
placed in individual 600 mL beakers and given a 10 min acclimation period. During
conditioning sessions, pups received 11 pairings of a 30 sec peppermint odor with a 0.5
mA hindlimb shock during the last 1 sec of odor, with an intertrial interval (ITI) of 4 min.
The odor (peppermint, McCormick & Co Inc.) was delivered by a flow dilution
olfactometer (2 liters/min flow rate) at a concentration of 1:10 peppermint to air vapor.
Control group. The mother and her pups were housed in a cage with abundant (57 cm layer) nesting/bedding material from PN8-12, during which time they were not
disturbed. This environment permits the mother to build a nest and spend most of her
time inside the nest caring for pups (Table 1).
Infant Y-maze test
At PN13, pups were assessed with a 5-trial Y-maze (start box: 8.5 x 10 X 8 cm;
choice arms: 8.5 x 24 x 8 cm) to measure approach responses to the natural maternal odor
or conditioned peppermint odor. After 5 sec in the start box, the alley doors were opened
and pups were given 60 sec to choose an arm. A response was considered a choice when
a pup’s entire body moved past the entrance to the alley.
Testing conditions included:
1) Abusive mother: Pups that were reared by an abusive mother were given the
choice of natural maternal odor (anesthetized mother) or the familiar odor (20 mL
of clean shavings).
2) Odor-shock: Pups that were reared by a control mother and that received daily
peppermint odor-shock conditioning from PN8-12 were given the choice of the
conditioned peppermint odor (20 µL peppermint on a Kim-wipe) or the familiar
odor (20 mL of clean shavings).
3) Control: Pups that were reared by a control mother but that did not receive odorshock conditioning were given the choice of natural maternal odor (anesthetized
mother) or the familiar odor (20 mL of clean shavings).
4) Neutral odor: Pups that were reared by a control mother but that did not receive
odor-shock conditioning were given the choice of the conditioned peppermint
odor (20 µL peppermint on a Kim-wipe, no value) or the familiar odor (20 mL of
clean shavings). The only difference between this group and the control group is
that the animals in the control group were tested using the natural maternal odor.
For this neutral odor group, the peppermint has not acquired the value of maternal
odor, as the animals did not encounter the peppermint odor in infancy.
Adult behavioral tests
All animals were tested in adulthood (≥ PN70) either with or without continuous
presentation of the natural maternal odor (for control and abused animals) or peppermintconditioned maternal odor (for odor-shock conditioned animals). For the natural maternal
odor, two anesthetized mothers were placed in an airtight glass jar (20 x 21 cm)
connected to a flow dilution olfactometer (10 liters/min flow rate) at the maximum
concentration of 1 odor:1 air. Because mothers eat the same diet and the maternal odor is
diet-dependent, pups cannot distinguish between their biological mother and a nonbiological mother. For the peppermint-conditioned maternal odor, the odor used during
infant conditioning (peppermint) was delivered by a flow dilution olfactometer (2
liters/min flow rate) at a concentration of 1:10 peppermint vapor.
Forced swim test (FST). Depressive-like behavior was tested in a tank (30 x 24 x
47.5 cm) filled with water (25±1°C) to achieve a depth that prevented escape and the tail
touching the bottom. Animals were habituated for 15 min one day prior to testing, and
tested for 5 min on the next day. The time the animal spent immobile (passive floating
without struggling, in a slightly hunched but upright position with minor movements
necessary to maintain the head above water) was recorded. During testing, a Plexiglas lid
covered the FST apparatus to ensure the odor did not dissipate.
Social behavior test. Social behavior was tested in a two-chamber apparatus (60 x
60 x 70 cm) built out of black Plexiglas. A black Plexiglas division (60 x 60 cm)
separated the two chambers and a square opening (15 x 13 cm) allowed animals to move
between chambers. Two metal cubes (6 x 6 x 6 cm) with holes (1 cm) on all sides and a
metal grid with 0.5 cm openings on the top were placed in each chamber during an
acclimation period of 5 minutes. After acclimation, a younger (PN 28-38) same sex
animal was placed inside one of the metal cubes and time spent by the experimental
animal in each chamber was recorded for 10 minutes.
Sexual motivation test. Sexual motivation was assessed in a Plexiglas test box (26
x 50 x 30 cm) for 10 min. The tests were performed under red lights during the early part
of the dark cycle. The adult male was given a 10 min adaptation period to the enclosure
prior to introducing the sexually receptive female rat of similar age, and the number of
mounts was recorded.
Adult amygdala local field potential (LFP) recordings
In a separate set of adult animals that were not run on the above behavioral tests
but were exposed to the same early-life manipulations, LFPs were obtained from the
amygdala in response to odor presentations. All animals were tested with both the odor of
an anesthetized lactating female and with peppermint.
Surgical Procedures. Animals were anesthetized with isoflurane and placed in a
stereotaxic apparatus using aseptic conditions. The scalp was reflected and a hole was
drilled in the skull for the recording electrode using coordinates to target the amygdala
complex (2.3 mm posterior from bregma; 5.0 laterally over the left hemisphere). A
bipolar teflon coated 0.18 mm diameter stainless steel electrode was lowered (8.0 mm
ventral from surface of the brain) and dental cement was placed over the hole to hold the
electrode in place. The electrode was connected to a telemetry pack (DSI) inserted
subcutaneously on the animal’s back. Topical lidocaine hydrochloride jelly (2% Akorn)
was applied and the incision sutured. Upon waking, animals were placed back into their
home cage that was placed on a heating pad for 30 minutes to 1 hour until observed to be
fully recovered and mobile. Animals were given 1 week for recovery before LFP
recording sessions.
LFP Recordings. A detailed description of the experimental procedures and
analysis can be found in Sarro et al, 2014. Briefly, LFP activity in response to the natural
or peppermint-conditioned maternal odor was recorded in the freely behaving rats. For a
typical recording section, the experimental animal was placed in a small cage in a sound
attenuated recording booth and amygdala LFP activity was recorded continuously
throughout a 5 min habituation period, followed by 5 presentations of a 5 sec odor with 5
min ITIs. Each animal was subjected to 2 recording sessions during the day: in one
session, the animal was presented with the natural maternal odor (using anesthetized
mothers, as described above) and in the other session, the animal was presented with
peppermint-conditioned maternal odor (learned in the infant odor-shock conditioning).
The inter-session interval was at least 3 hr and animals were placed in novel cages for
each of the two sessions. All odors were presented using a flow-dilution olfactometer
(same concentration/procedure used in the behavioral tests) and solenoid that allowed the
odor to flow for a total of 5 sec during each trial. Odors were not matched for intensity,
but were both easily detectable by humans. Stimulus onset was noted online and used to
independently assess each response offline. Neural signals were amplified, filtered (0.5 to
300 Hz) digitized at 2 kHz with Spike2 software (CED, Inc) and analyzed offline.
Data analysis. Fast Fourier Transform (FFT) power analyses were performed on
the raw LFP data in intervals taken directly from portions of each session’s neural trace
that corresponded with periods of time immediately before and during each odor
presentation, to quantify LFP oscillatory power in 2.4 Hz frequency bins from 0–100 Hz
(Hanning). Power in the theta (5–15 Hz), beta (15-35 Hz) and gamma (35-90 Hz)
frequency bands was calculated for each specified window. Odor-evoked change in LFP
oscillatory power was calculated as percent change in power from the baseline activity
during the 5 sec before the odor presentation to 5 sec during the odor presentation in each
frequency band. Repeated measures ANOVA’s were run to test for main effects of odor
type on odor-evoked response, followed by post hoc analyses to examine differences
between specific LFP frequencies (ANOVA’s or t-tests to compare specific frequency
bins).
Verifying electrode placement. Following the recording sessions, animals were
anesthetized (urethane), perfused (0.9% saline followed by 10% formaldehyde), brains
were removed and stored in a 30% sucrose/10% formaldehyde solution for later
sectioning. The brains were then sectioned coronally (40 μm), mounted on subbed slides,
and stained with cresyl violet. Electrode tracks and recording locations were verified
under a light microscope, and images were acquired using a digital camera.
Statistical analysis
All data were expressed as mean ( SEM). The maternal behavior, pups’ behavior
and body weight data (Table 1) were analyzed using Student’s t-test. The Y-maze data
were analyzed by one-way ANOVA followed by post hoc Fisher tests. The adult FST,
social behavior, and sexual motivation data were analyzed by two-way ANOVA (infant
condition and maternal odor presentation as factors) followed by post hoc Fisher tests. In
all cases, differences were considered significant when P < 0.05.
3. Supplementary results
Infant odor-shock conditioning and adult sexual motivation.
Early-life experiences profoundly affect adult behaviors that are related to sexual
function. For example, clinical studies show that humans with abusive childhood
experiences are more likely to show early pubertal onset and precocious sexuality and, in
adulthood, to get involved with an adult abusive relationship and provide limited
investment in child rearing (Belsky et al, 1991; Delsol and Margolin, 2004; Messman and
Long, 1996; Messaman-Moore and Long, 2003; Taft et al, 2008). Animal models, such as
those involving neonatal handling (Padoin et al, 2001; Raineki et al, 2013) and variations
in maternal licking of pups (Cameron et al, 2008; Uriarte et al, 2007), suggest long-term
changes in sexual behavior and motivation as a result of these early-life experiences.
However, the effect of animal models of abusive attachment on sexual function has not
been investigated.
Interestingly, it has been shown that odors experienced in a mother-infant
attachment context can retain their value into adulthood and modulate various behavioral
systems, including those supporting reproduction (Fillion and Blass, 1986; Moore et al,
1996) and food choice (Galef and Heiber, 1976; Leon et al, 1977; Sevelinges et al, 2009).
This phenomenon is not limited to typical attachment: results from our laboratory have
shown that the conditioned odor learned in an abusive attachment paradigm (infant odorshock conditioning) is capable of attenuating fear learning and amygdala activity
(Sevelinges et al, 2007; Moriceau et al, 2009) and rescues depressive-like behaviors
(Sevelinges et al, 2011). However, the modulation of sexual motivation by cues
associated with an abusive attachment has not been analyzed. Here we assessed the
potential effects of an abusive attachment odor on adult male sexual behavior.
Infant olfactory classical conditioning paradigm. Beginning at PN8, pups were
odor-shock conditioned daily for 5 consecutive days. Pups were removed from their
mother, which stayed in the home cage, and were transferred to a different room where
they were placed in individual 600 mL beakers and given a 10 min acclimation period.
During conditioning sessions, pups received 11 presentations of a 30 sec peppermint odor
and a 0.5 mA hindlimb shock, with an intertrial interval of 4 min. The odor (peppermint,
McCormick & Co Inc.) was delivered by a flow dilution olfactometer (2 liters/min flow
rate) at a concentration of 1:10 peppermint to air vapor.
1) Paired odor-shock pups received 11 pairings of the 30 sec odor with shock
overlapping during the last 1 sec of the odor presentation.
2) Unpaired odor-shock pups received the shock 2 min after each 30 sec odor
presentation.
3) Odor-only pups received only the peppermint odor presentations
Sexual motivation test. All animals were tested in adulthood (≥ PN70) either with
or without continuous presentation of the conditioned peppermint odor in a Plexiglas test
box (26 x 50 x 30 cm) for 10 min. The peppermint odor that was used during infant
conditioning was delivered by a flow dilution olfactometer (2 liters/min flow rate) at a
concentration of 1:10 peppermint vapor. The tests were performed under red lights during
the early part of the dark cycle. The adult male was given a 10 min adaptation period to
the enclosure prior to introducing the sexually receptive female rat of similar age, and the
number of mounts was recorded. Results were analyzed using two-way ANOVA (infant
condition and maternal odor presentation as factors) followed by post hoc Fisher tests. In
all cases, differences were considered significant when P ≤ 0.05.
Results and Discussion
As shown in Supplemetary Figure 1, none of the infant treatments (paired,
unpaired, and odor-only conditions) were able to induce changes in the number of mounts
in adulthood. However, the presence of the odor experienced in infancy (peppermint)
increased the number of mounts only in the animals that received paired odor-0.5mA
shock conditioning (abusive attachment) in infancy (infant conditioning x odor
presentation ANOVA F(2,33) = 3.146, p<0.05; post hoc fisher tests revealed paired odor–
0.05mA shock conditioned animals tested in the presence of the CS odor significantly
differ from all other groups). These results indicate that the odor must be learned in
infancy to be able to modulate sexual motivation in adulthood. Moreover, the ability of
the odor learned in infancy to modulate adult behaviors is enduring even when paired
with aversive events.
Number of mounts
Sexual Motivation
18
15
*
No odor
Peppermint odor
12
9
6
3
0
Paired
Unpaired Odor only
Supplementary Figure 1. Adult sexual motivation test with or without the continuous
presentation of the peppermint odor used in infant odor-shock conditioning. Neither of
the infant odor-shock conditions (paired, unpaired) induced deficits in the number of
mounts when compared to control rats (odor only); however, conditioned peppermint
odor presentation increased the number of mounts only in paired odor-shock animals. * P
< 0.05, significant difference from each group (n=5-8 per group).
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