1. Supplementary background

Maternal odor attachment learning

In order for an infant rat pup to survive, it must learn to identify its mother’s diet-

dependent 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 mother-

infant 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 long-

lasting 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 (201°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 odor-

shock 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 (5-

7 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 odor-

shock 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 peppermint-

conditioned 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 non-

biological 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 odor-

shock 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.

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).

Supplementary References

Baldauf D, Desimone R. (2014). Neural mechanisms of object-based attention. Science

344: 424-427.

Bauer EP, Paz R, Paré D (2007). Gamma oscillations coordinate amigdalo-rhinal

interactions during learning. J Neurosci 27: 9369-9379.

Belsky J, Steinberg L, Draper P (1991). Childhood experience, interpersonal

development, and reproductive strategy: An evolutionary theory of socialization.

Child Develop 62: 647-670.

Brake SC (1981). Suckling infant rats learn a preference for a novel olfactory stimulus

paired with milk delivery. Science 211: 506-508.

Buzsáki G (2006). Rhythms of the brain. Oxford University Press: New York, NY.

Cameron N, Del Corpo A, Diorio J, McAllister K, Sharma S, Meaney MJ (2008).

Maternal programming of sexual behavior and hypothalamic-pituitary-gonadal

function in the female rat. PLoS One 3: e2210.

Camp LL, Rudy JW (1988). Changes in the categorization of appetitive and aversive

events during postnatal development of the rat. Dev Psychobiol 21: 25-42.

Cardin, JA, Carlén M, Meletis K, Knoblich U, Zhang F, Deisseroth K, Tsai LH, Moore

CI (2009). Driving fast-spiking cells induces gamma rhythm and controls sensory

responses. Nature 459: 663-668.

Diekelmann S, Born J (2010). The memory function of sleep. Nature Rev 11: 114-126.

Delsol C, Margolin G (2004). The role of family-of-origin violence in men’s marital

violence perpetration. Clin Psychol Rev 24: 99-122.

Ehrlich DE, Ryan SJ, Hazra R, Guo JD, Rainnie DG (2013). Postnatal maturation of

GABAergic transmission in the rat basolateral amygdala. J Neurophysiol 110: 926-

941.

Engel AK, Fries P, Singer W (2001). Dynamic predictions: oscillations and synchrony in

top-down processing. Nature Rev 2: 704-716.

Fanselow EE, Nicolelis MA (1999). Behavioral modulation of tactile responses in the rat

somatosensory system. J Neurosci 19: 7603-7616.

Fillion TJ, Blass EM (1986). Infantile experience with suckling odors determines adult

sexual behavior in male rats. Science 231: 729-731.

Galef Jr BG, Heiber L (1976). Role of residual olfactory cues in the determination of

feeding site selection exploration patterns of domestic rats. J Comp Physiol Psychol

90: 727-739.

Galef Jr BG, Kaner HC (1980). Establishment and maintenance of preference for natural

and artificial olfactory stimuli in juvenile rats. J Comp Physiol Psychol 94: 588-595.

Galef Jr BG, Sherry DF (1973). Mother’s milk: a medium for transmission of cues

reflecting the flavor of mother’s diet. J Comp Physiol Psychol 83: 374-378.

Gervasoni D, Lin S, Ribeiro S, Soares ES, Pantoja J, Nicolelis MAL (2004). Global

forebrain dynamics predict rat behavioral states and their transitions. J Neurosci 24:

11137-11147.

Haroutunian V, Campbell BA (1979). Emergence of interoceptive and exteroceptive

control of behavior in rats. Science 205: 927-929.

Headley DB, Weinberger NM (2013). Fear conditioning enhances γ oscillations and their

entrainment of neurons representing the conditioned stimulus. J Neurosci 33: 5705-

5717.

Hill KT, Warren M, Roth TL (2014). The influence of infant-caregiver experiences on

amygdala Bdnf, OXTr, and NPY expression in developing and adult male and female

rats. Behav Brain Res 22: 175-280.

Hofer MA, Shair H, Singh P (1976). Evidence that maternal ventral skin substances

promote suckling in infant rats. Physiol Behav 17: 131-136.

Hofer MA, Sullivan RM (2001). Toward a neurobiology of attachment. In: Nelson CA,

Lucian M (eds). Developmental cognitive neuroscience. MIT Press: Cambridge, MA,

pp 5 99-616.

Ivy AS, Brunson KL, Sandman C, Baram TZ (2008). Dysfunctional nurturing behavior in

rat dams with limited access to nesting material: a clinically relevant model for early-

life stress. Neuroscience 154: 1132-1142.

Johanson IB, Teicher MH (1980). Classical conditioning of an odor preference in 3-day-

old rats. Behav Neural Biol 29: 132-136.

Jensen O, Kaiser J, Lachaux JP (2007). Human gamma-frequency oscillations associated

with attention and memory. Trends Neurosci 30: 317-324.

Landers MS, Sullivan RM (2012). The development and neurobiology of infant

attachment and fear. Dev Neuroci 34: 101-114.

Lasztóczi B, Klausberger T (2014). Layer-Specific GABAergic Control of Distinct

Gamma Oscillations in the CA1 Hippocampus. Neuron 81: 1126-1139.

Leon M (1975). Dietary control of maternal pheromone in the lactating rat. Physiol

Behav 14: 311-319.

Leon M (1992). Neuroethology of olfactory preference development. J Neurobiol 23:

1557-1573.

Leon M, Galef BG, Behse JH (1977). Establishment of pheromonal bonds and diet choice

in young rats by odor pre-exposure. Physiol Behav 18: 387-391.

Logan DW, Brunet LJ, Webb WR, Cutforth T, Ngai J, Stowers L (2012). Learned

recognition of maternal signature dos mediates the first suckling episode in mice.

Curr Biol 22: 1998-2007.

Messman TL, Long PJ (1996). Child sexual abuse and its relationship to revictimization

in adult women: A review. Clin Psychol Rev 16: 397-420.

Messman-Moore TL, Long PJ (2003). The role of childhood sexual abuse sequelae in the

sexual revictimization of women: An empirical review and theoretical reformulation.

Clin Psychol Rev 23: 537-571.

Moore CL, Jordan L, Wong L (1996). Early olfactory experience, novelty, and choice of

sexual partner by male rats. Physiol Behav 60: 1361-1367.

Moriceau S, Raineki C, Holman JD, Holman JG, Sullivan RM (2009). Enduring

neurobehavioral effects of early-life trauma mediated through learning and

corticosterone suppression. Front Behav Neurosci 3: 22.

Moriceau S, Sullivan RM (2006). Maternal presence serves as a switch between learning

fear and attraction in infancy. Nat Neurosci 9: 1004-1006.

Murthy VN, Fetz EE (1992) Coherent 25- to 35-Hz oscillations in the sensorimotor

cortex of awake behaving monkeys. Proc Natl Acad Sci USA 89: 5670-5674.

Nicolelis MA, Baccala LA, Lin RC, Chapin JK (1995). Sensorimotor encoding by

synchronous neural ensemble activity at multiple levels of the somatosensory system.

Science 268: 1353-1358.

Padoin MJ, Cadore LP, Gomes CM, Barros HMT, Lucion AB (2001). Long-lasting

effects of neonatal stimulation on the behavior of rats. Behav Neurosci 115: 1332-

1340.

Pedersen PE, Williams CL, Blass EM (1982). Activation and odor conditioning of

suckling behavior in 3-day-old albino rats. J Exp Psychol Anim Behav Process 8:

329-341.

Raineki C, Lutz ML, Sebben V, Ribeiro RA, Lucion A (2013). Neonatal handling

induces deficits in infant mother preference and adult partner preference. Dev

Psychobiol 55: 496-507.

Raineki C, Moriceau S, Sullivan RM (2010). Developing a neurobehavioral animal

model of infant attachment to an abusive caregiver. Biol Psychiatry 67: 1137-1145.

Raineki C, Rincón-Cortés M, Belnoue L, Sullivan RM (2012). Effects of early-life abuse

differ across development: Infant social behavior deficits are followed by adolescent

depressive-like behaviors mediated by the amygdala. J Neurosci 32: 7758-7765.

Ravel N, Chabaud P, Martin C, Gaveau V, Hugues E, Tallon-Baudry C, Bertrand O,

Gervais R (2003). Olfactory learning modifies the expression of odour-induced

oscillatory responses in the gamma (60–90 Hz) and beta (15–40 Hz) bands in the rat

olfactory bulb. Eur J Neurosci 17: 350-358.

Roth TL, Sullivan RM (2005). Memory of early maltreatment: neonatal behavioral and

neural correlates of maternal maltreatment within the context of classical

conditioning. Biol Psychiatry 57: 823-831.

Sanes JN, Donoghue JP (1993). Oscillations in local field potentials of the primate motor

cortex during voluntary movement. Proc Natl Acad Sci USA 90: 4470-4474.

Sarro EC, Wilson DA, Sullivan RM (2014). Maternal regulation of infant brain state.

Curr Biol 24: 1664-1669.

Sevelinges Y, Levy F, Mouly AM, Ferreira G (2009). Rearing with artificially scented

mothers attenuates conditioned odor aversion on adulthood but not its amygdala

dependency. Behav Brain Res 198: 313-320.

Sevelinges Y, Moriceau S, Holman P, Miner C, Muzny K, Gervais R, Mouly AM,

Sullivan RM (2007). Enduring effects of infant memories: Infant odor-shock

conditioning attenuates amygdala activity and adult fear conditioning. Biol Psychiatry

62: 1070-1079.

Sevelinges Y, Mouly AM, Raineki C, Moriceau S, Forest C, Sullivan RM (2011). Adult

depression-like behavior, amygdala and olfactory cortex functions are restored by

odor previously paired with shock during infant’s sensitive period attachment

learning. Dev Cog Neurosci 1: 77-87.

Sinfield JL, Collins DR (2006). Induction of synchronous oscillatory activity in the rat

lateral amygdala in vitro is dependent on gap junction activity. Eur J Neurosci 24:

3091-3095.

Steriade M, McCormick DA, Sejnowski TJ (1993). Thalamocortical oscillations in the

sleeping and aroused brain. Science 262: 679-685.

Sullivan RM, Landers M, Yeamen B, Wilson DA (2000a). Good memories of bad events

in infancy. Nature 407: 38-39.

Sullivan RM, Stackenwalt G, Nasr F, Lemon C, Wilson DA (2000b). Association of an

odor with activation of olfactory bulb noradrenergic B-receptors or locus coeruleus

stimulation is sufficient to produce learned approach response to that odor in neonatal

rats. Behav Neurosci 114: 957-962.

Sullivan RM, Wilson DA, Wong R, Correa A, Leon M (1990). Modified behavioral and

olfactory bulb responses to maternal odors in preweanling rats. Dev Brain Res 53:

243-247.

Taft CT, Schumm JA, Marshall AD, Panuzio J, Holtzworth-Munroe A (2008). Family-of-

origin maltreatment, posttraumatic stress disorder symptoms, social information

processing deficits, and relationship abuse perpetration. J Abnorm Psychol 117: 637-

646.

Teicher MH, Blass EM (1977). First suckling response of the newborn albino rat: the

roles of olfaction and amniotic fluid. Science 198: 635-636.

Teicher MH, Flaum LE, Williams M, Eckhert SJ, Lumia AR (1978). Survival, growth,

and suckling behavior or neonatally bulbectomized rats. Physiol Behav 21: 553-561.

Thompson JV, Sullivan RM, Wilson DA (2008). Developmental emergence of fear

learning corresponds with changes in amygdala synaptic plasticity. Brain Res 1200:

58-65.

Tononi G (2009). Slow wave homeostasis and synaptic plasticity. J Clin. Sleep Med 5:

S16-S19.

Tononi G, Cirelli C (2006). Sleep function and synaptic homeostasis. Sleep Med Rev 10:

49-62.

Traub RD, Whittington MA, Colling SB, Buzsáki G, Jefferys JG (1996). Analysis of

gamma rhythms in the rat hippocampus in vitro and in vivo. J Physiol 493: 471-484.

Tyler K, Moriceau S, Sullivan RM (2007). Long-term colonic hypersensitivity in adult

rats induced by neonatal unpredictable vs. predictable shock. Neurogastroenterol

Motil 19: 761-768.

Uriarte N, Breigeiron MK, Benetti F, Rosa XF, Lucion AB (2007). Effects of maternal

care on the development, emotionality and reproductive functions in male and female

rats. Dev Psychobiol 49: 451-462.

Vanderwolf CH (1969). Hippocampal electrical activity and voluntary movement in the

rat. Electroencephalogr Clin Neurophysiol 26: 407-418.

Volman V, Behrens MM, Sejnowski TJ (2011). Downregulation of parvalbumin at

cortical GABA synapses reduces network gamma oscillatory activity. J Neurosci 31:

18137-18148.

Wilson DA, Sullivan RM (1994). Neurobiology of associative learning in the neonate:

early olfactory learning. Behav Neural Biol 61: 1-18.