Developmental Psychobiology

Rosario Montirosso1
Livio Provenzi1
Ed Tronick2
Francesco Morandi3
Gianluigi Reni4
Renato Borgatti5
1

03 Center for the Study of Social

Emotional Development of at Risk Infant
Scientific Institute
IRCCS Eugenio Medea
Bosisio Parini, LC, Italy
E-mail: rosario.montirosso@bp.lnf.it
2
Department of Psychology
University of Massachusetts
Boston, MA
3
Pediatric Unit
Sacra Famiglia Hospital
Erba, CO, Italy
4

Bioengineering Laboratory
Scientific Institute
IRCCS Eugenio Medea
Bosisio Parini, LC, Italy

Department of Child and Adolescent

Neurology and Psychiatry
Scientific Institute
IRCCS Eugenio Medea
Bosisio Parini, LC, Italy

Vagal Tone as a Biomarker of

Long-Term Memory for a
Stressful Social Event at
4 Months
ABSTRACT: This study investigates infants memory for social stress after a
15-day retention interval using behavior and respiratory sinus arrhythmia (RSA).
Experimental group infants were exposed to face-to-face still-face paradigm
(FFSF) two times; the first time at 4 months and after a 2-week interval.
Control group infants were exposed to FFSF only once at 4 months plus 2 weeks.
Infants were categorized as suppressors or non-suppressors based on the
direction of RSA change at first FFSF exposure. No behavioral differences were
found among groups and exposure conditions. In the experimental group
suppressors changed and showed no suppression when re-exposed 2 weeks later
to FFSF. Non-suppressors showed no change in RSA from the first to the second
exposure to FFSF. Control infants showed similar RSA changes to experimental
infants at their first exposure. Findings indicate that 4-month-old infants have
memory for social stress related to individual differences in autonomic reactivity.
2014 Wiley Periodicals, Inc. Dev Psychobiol
Keywords: vagal tone; infants; memory; physiological reactivity; social stress;
still-face paradigm

An extensive literature has documented that memory

capacity emerges early in the first year of life in human
infants (Bauer, 2013; Rose, Feldman & Jankowski,
2001; Rovee-Collier & Gilles, 2010). Young infants
showed operant conditioning during a mobile conjugate
reinforcement experiment, with a 1 day retention
time at 3-months and up to 7 days at 3-months (cf.
Hartshorn, Rovee-Collier, Gerhardstein, Bhatt, Wondoloski, Klein et al., 1998; Rovee-Collier, Hartshorn, &
DiRubbo, 1999). A study adopting the visual paired
Manuscript Received: 14 February 2014
Manuscript Accepted: 30 July 2014
Correspondence to: Rosario Montirosso
Contract grant sponsor: Italian Health Ministery (Ricerca Corrente
2007 Sviluppo dei processi di attenzione e di memoria in neonati
sani e nati in condizioni di rischio evolutivo)
Contract grant sponsor: NICHD
Contract grant number: 5R01HD050459
Contract grant sponsor: NSF
Contract grant number: 0819839
Article first published online in Wiley Online Library
(wileyonlinelibrary.com).
DOI 10.1002/dev.21251
2014 Wiley Periodicals, Inc.

comparison task documented that that 3- and 6-montholds remembered faces after a 24 hr retention interval
(Pascalis, de Haan, Nelson, & de Schonen, 1998).
Using a deferred imitation task 9-month-olds recall the
individual actions of multistep sequences, even if only
45% of the infants evidenced recall sequences in the
correct order after 1 month (Bauer, Wiebe, Waters, &
Bangston, 2001). Taken together, these findings on
typically-developing infants converge to suggest that
during the first months of life long-term memory
capacity increases.
Nevertheless, research to date has been primarily
focused on neutral events and non-social cognition,
such as learning tasks (Rovee-Collier & Cuevas, 2009)
and deferred imitation (Carver & Bauer, 2001), whereas
the memory for social stress events has received far
less attention (Haley, 2013). One study demonstrated
that 6-month-old infants showed an anticipatory
response associated with a moderate social stress seen
24 hr earlier (Haley, Cordick, Mackrell, Antony, &
Ryan-Harrison, 2010a). Recent findings from our group,

Montirosso et al.

adopting salivary cortisol reactivity as measure of stress

and memory in a different cohort of 4-month-old
infants, highlighted a memory for a brief relational
disruption of maternal communication after a 15-day
retention interval, when the infants were again exposed
to same social event (Montirosso, Tronick, Morandi,
Ciceri, & Borgatti, 2013). Bornstein, Arterberry and
Mash (2004) found evidence suggestive of memory for
a mildly negative emotional event experienced at
5 months of age in 20-month-olds. The scarcity of
research on infants memory for social stress is
surprising given the view that infants early social and
stressful experiences, including their early interactions,
contribute critically to socio-emotional development
and memory capacities (Dawson, Ashman, & Carver,
2000; DiCorcia & Tronick, 2011). In the current study,
using behavioral responses and vagal tone, we examined whether 4-month-old infants memory for a
stressful social event (i.e., maternal still-face) was
preserved after a 15-day retention interval when they
were again exposed to same event. To evaluate if there
was a memory for social stress, we compared the
infants responses in the experimental group to those of
age-matched control infants with no prior exposure to
the same stressful event.
Vagal tone is an autonomic measure of parasympathetic activity, which is assumed to be regulated by the
nucleus ambiguus in the brain stem. It is thought to be
an index of neural regulation of the cardiac activity via
the vagus nerve (Porges, 2011). A widely adopted
measure of the vagal tone is the respiratory sinus
arrhythmia (RSA), which reflects the fluctuations in the
respiratory component of the inter-beat interval of the
heart rate (Staton, El-Sheikh, & Buckhalt, 2009). Vagal
tone inhibits sympathetic arousal, promotes a more
stable behavioral state, and supports social engagement
(Porges, 2011; Porges, Doussard-Roosevelt, Portales, &
Greenspan, 1996). Under stressful conditions, parasympathetic withdrawal, or a decrease in RSA relative to
baseline, allows for sympathetic activation and mobilization of the organism to respond to the stressor
(Hofheimer, Wood, Porges, Pearson, & Lawson, 1995).
Thus, higher basal vagal tone and vagal brake withdrawal in response to a stressor are considered adaptive
and have been associated with better state regulation,
more attentional control, and greater social competence
(Suess, Porges, & Plude, 1994; Stifter & Corey, 2001).
Previous studies have found a relation between vagal
tone and memory processes (Backs, Ryan, & Wilson,
1994). In an animal model, a gastrointestinal hormone
(i.e., cholecystokinin-octapeptide), which is released
during feeding, has a memory-enhancing effect when
administered intraperitoneally, but not in vagotomized
mice. Thus, this gastrointestinal hormone seems to

Developmental Psychobiology

produce its effect on memory by activating ascending

fibers in the vagus nerve (Flood, Smith, & Morley,
1987). In human adults, vagus nerve stimulation was
associated with better retention in a word-recognition
memory task (Clark, Naritoku, Smith, Browning, &
Jensen, 1999). In children few studies have examined
the link between RSA withdrawal and memory, and
results are mixed. For instance, in preschoolers greater
parasympathetic withdrawal was related to better memory performance (Quas, Bauer, & Boyce, 2004),
however, in a subsequent study the same researchers
did not find that association (Quas, Carrick, Alkon,
Goldstein, & Boyce, 2006). In 6-month-old infants,
vagal tone was associated with better performance
during a visual recognition memory task (i.e., infants
looked longer at the novel stimulus than the familiar
stimulus; Linnemeyer & Porges, 1986). Using a deferred imitation task, Haley, Grunau, Weinberg, Keidar
& Oberlander (2010b) found that greater suppression of
vagal tone during the task demonstration was related to
better memory recall in 6-month-old infants. Overall,
these results support the idea that vagal activity plays a
role both in the memory consolidation and recall for
non-social tasks. Nevertheless, to the best of our
knowledge, the research on the role of vagal tone in
infant memory for a stressful social event has been
neglected.
The face-to-face-still-face (FFSF; Tronick, Als,
Adamson, Wise, & Brazelton, 1978) procedure is a
well-established paradigm, which involves a perturbation in maternal communication that is behaviorally
and physiologically stressful for young infants (Mesman, van IJzendoorn, & Bakermans-Kranenburg,
2009). During the FFSF the infant is confronted first
with a brief period of normal motherinfant face-toface interaction (the Play episode), followed by 2 min
of the mothers neutral and unresponsive face (the StillFace episode), and ending with infant and mother
resuming normal play (the Reunion episode). Infant
responses to the mothers lack of responsivity are
marked by increases in negative affect, a drop in
positive affect and in social monitoring (for reviews
see, Adamson & Frick, 2003; Mesman et al., 2009).
Vagal activity has also been measured in the context of
FFSF. Consistent with the evidence that vagal tone is
reduced during challenging situations, FFSF studies
have found a decrease in RSA from baseline (Bazhenova, Plonskaia, & Porges, 2001; Moore & Calkins,
2004) or from the play (Weinberg & Tronick, 1996;
Conradt & Ablow, 2010) to the Still-Face episode,
indicating that infants are attempting to cope with the
stressful effects of maternal non-responsiveness. Nonetheless, research has also documented individual differences in vagal reactivity with some infants exhibiting

Developmental Psychobiology

the expected vagal brake withdrawal in response to

maternal unresponsiveness (so called, suppressors),
whereas others (so called, non-suppressors) did not
give evidence of the same pattern (Moore & Calkins,
2004). In a study using the FFSF paradigm, 5-monthold infants were divided in suppressor and non-suppressor based on their vagal activity response to the stillface (Bazhenova et al., 2001). Infants who did not
exhibit a decrease in vagal tone to the maternal stillface appeared to be more emotionally negative.
Although, difficulty in the ability to suppress RSA
during a social challenge may be related to a lack of
behavioral and emotional control (Porges, 1996; Wilson
& Gottman, 1996), the question of how individual
differences in RSA are related to infant memory for
emotional events, such as FFSF, remains open.
In infant studies adopting the FFSF paradigm, both
cortisol and cardiac activity have been utilized as
physiological indexes of stress. However, findings
indicated only limited associations between these
measures. A correlation between cortisol reactivity and
vagal tone was found during the maternal still-face, but
not during the reunion episode (Feldman, Singer &
Zagoory, 2010). No significant correlation emerged
between cardiac activity and poststress salivary cortisol
in a sample of 5- and 6-month-olds infants during a
modified FFSF procedure that used an additional stillface reunion sequence (Haley and Stansbury, 2003).
The lack of association may not be surprising given
that stress reactivity is not a unitary process (Boyce
and Ellis, 2005). Salivary cortisol is a measure of
activity of the hypothalamicpituitaryadrenal (HPA)
axis and provides a global measure of poststress
reactivity. The RSA is a measure of parasympathetic
activity, which allows for an examination of the
ongoing stress response across the FFSF.
Consequently, given that cortisol and RSA represent
different physiological systems, as a complement to our
previous study on HPA and memory for social stress, in
the present report we examine the possibility that vagal
reactivity is related to infants memory for social stress.
We employed the same design adopted in the HPA
study (Montirosso et al., 2013), with a separate cohort
of infants, using multiple measures of memory including behavioral response (i.e., negative emotionality
across the FFSF) and RSA. We compared an experimental group of infants memory for maternal unavailability after a 15-day retention interval to that of
age-matched control group of infants with no prior
exposure to the FFSF, taking into account individual
differences in vagal tone activity. We expected that
infants in the experimental condition would exhibit
evidence for memory for the FFSF at physiological
level compared to age-matched control infants. Impor-

Vagal Tone and Early Memory for Social Stress

tantly, we evaluated how individual differences in

infants physiologic response (i.e., suppressors or nonsuppressors) at the first exposure to social stress related
to memory for the FFSF at the second exposure,
15 days later. Though tentative because of a lack of
previous studies on RSA associated with infant social
memory, we expected a memory effect, which would
be affected by individual differences in stress reactivity.
That is, if some infants evidenced a pronounced
suppression to the maternal still-face (i.e., suppressors),
while others respond with an increase of the RSA (i.e.,
non-suppressors), we expected that their vagal tone
would also differ from the first exposure to the second
one on the basis of these individual differences. Given
the our previous findings of a lack of behavioral
differences (Montirosso et al., 2013), we did not expect
that the experimental infants to show a difference in
the behavioral engagement (e.g., more or less infant
negativity) during their second exposure to the FFSF
compared to their first exposure, or compared to the
age-matched control infants.

METHODS
Participants
Eighty-three 4-month-olds infants and their mothers participated in the study. The infants were all healthy full-terms
recruited from the regular nursery at Pediatric Unit of the
Sacra Famiglia Hospital of Erba (Como), Italy. The selection
criteria for the infants were: full-term gestation (37 weeks),
Apgar scores of at least 7 at 1 min and 8 at 5 min, no
congenital abnormalities, appropriate weight for gestational
age, and uncomplicated prenatal, perinatal and neonatal
courses.
The dyads were randomly assigned to the experimental or
control condition. For infants in the experimental condition
(N 40) the first exposure to the FFSF took place when the
infants were 4 months of age (T1-Exp) and the second
exposure was 2 weeks later (T2-Exp). Infants in the control
condition (N 43) were videotaped only once (T2-Ctrl), at
the age corresponding to the second exposure for infants in
the experimental conditions. The inclusion of a control group
insured that any changes in infants autonomic reactivity
between the first and second exposure would be a function of
the prior emotional stress due to the maternal unresponsiveness rather than the effect of the novelty of the laboratory or
other confounds at T1 and T2.
Procedure
All mothers were told that the study concerned infant
caregiver interactive behavior. The mothers of infants in the
experimental condition were also told the study was concerned with memory. Mothers who expressed an interest in
participating in the study were scheduled to visit the
laboratory of the Scientific Institute IRCCS Eugenio Medea

Montirosso et al.

when they thought their infant would be awake and alert. To

accommodate the schedules it was necessary for the testing to
be done throughout the day (9:30 a.m. to 17:00 p.m.). Mothers
completed a socio-demographic survey (i.e., infant demographic variables, obstetric characteristics and maternal characteristics) and socio-economic status was obtained according
to Hollingshead (1975) classification. Following the completion of the questionnaires a video recording of the mother
infant interaction was made using the FFSF procedure. The
study was approved by the Ethics Committee of the Scientific
Institute IRCCS Eugenio Medea and written informed consent
was obtained from every mother.

Developmental Psychobiology
FFSF. Averaged values were computed for the baseline, Play,
Still-Face, Reunion episodes. To examine changes in autonomic activity, ratio indexes of RSA relative to baseline
levels for each episode of the FFSF were calculated, by
dividing the average values of each FFSF episodes for the
baseline average values.
Consistent with previous studies (Moore & Calkins, 2004),
to examine individual differences in vagal tone during the first
exposure to the FFSF (i.e., T1-Exp and T2-Ctrl), infants were
assigned to suppressor or non-suppressor groups based on
whether they showed, respectively, a decrease or an increase
in RSA from the first normal interaction episode (i.e., Play)
to the Still-Face episode of the FFSF.

Laboratory Procedures. At each session mothers were

debriefed about the FFSF procedure. While infants were in a
quiet and alert state three disposable ECG electrodes were
placed in the usual inverted triangle pattern on the infants
chest. According to literature (Bazhenova et al., 2001; Moore
& Calkins, 2004), a 3-min period of baseline adaptation
preceded the FFSF to let the infant familiarize with the
laboratory environment together with the caregiver. During
this period, the mothers stayed near to the infant, but they
were instructed not to play with him/her nor to provide any
social stimulation. After the adaptation period, the FFSF
procedure began. It included three episodes: (1) a Play
episode: mothers and infants engaging in a normal face-toface interaction; (2) a Still-Face episode: mothers asked to
maintain a neutral face and to interrupt any communication
toward their infants; (3) a Reunion episode: mothers and
infants resumed their normal interaction. Additionally, mothers were asked to put on a yellow smock at the beginning of
the FFSF at each visit to serve as a unique memory cue and
to provide a distinctive memory cue for the infant at T2-Exp.
The smock was worn at both visits for the experimental
mothers and by the control group mothers at their single visit.

Physiological Data. ECG signal was collected throughout

the adaptation period and the FFSF procedure using the
ProComp Infiniti (Thought Technology, Ltd., Canada). The
output was transmitted as heart inter-beat intervals (IBI) to a
PC connected through i-Link IEEE1394 interface. ECG data
and on-line videotaping of the FFSF procedure were synchronized using BioGraph Infiniti (Thought Technology Ltd.,
Canada) PC software. IBI data were screened off-line for
artifacts due to infants movements.
Vagal Tone. The RSA estimation was obtained according
to Porgess (1985) method. Porgess algorithm uses a moving
21-point polynomial to detrend periodicities in heart periods
slower than RSA. As indicated by previous research (Huffman, Bryan, del Carmen, Pedersen, Doussard-Roosevelt, &
Porges, 1998; Stifter & Jain, 1996), a band-pass filter was
used to extract heart period variance within the frequency
band usually considered for young infants (0.241.04). The
estimate of RSA was obtained using the natural logarithm of
this variance and was expressed in units of ln(ms)2. RSA
were calculated on sequential 10-s epochs for the 1-min
baseline period and during each of the 2-min episodes of the

Missing Physiological Data. Six infants were excluded

from the present analysis. For one experimental group infant
RSA values across the FFSF were higher than 2 SDs from the
mean both during T1 and T2. ECG data were not available
for five infants (three in the Exp group and two in the Ctrl
group), due to technical reasons. Consequently, the final
sample included 36 infants in the experimental condition
(18 suppressors and 18 non-suppressors) and 41 infants in the
control group (17 suppressors and 24 non-suppressors).

Behavioral Data. The infants behavioral engagement was

micro-analytically coded using 1 s sampling by independent
coders masked to experimental condition with the Infant and
Caregiver Engagement Phases (ICEP; Weinberg & Tronick,
1998). In the present study a negative emotionality (e.g.,
sadness, distress, crying, or grimacing, complaining, being
fussy, crying vocalizations) index was calculated for each of
the five FFSF episodes. The index was the proportion
between the total time of negative emotionality per episode
by the total length of the episode. Coders were trained with a
gold standard sample of ten videotapes (agreement > 75%).
They were masked to group membership and unaware of the
cardiac data. Twenty percent of FFSF for all three sessions
were also randomly selected and evaluated for agreement
between two independent coders. Inter-observer reliability
was determined through percentage agreement and Cohens
kappa. The mean percentage of agreement and Cohens kappa
were 83% and .75, respectively. Negative emotionality data
for the whole sample did not meet the criteria for normal
distribution and were normalized using arcsine transformation
(Weinberg & Tronick, 1998).

Statistical Analysis. To evaluate if there were different

maternal and infant characteristics between experimental and
control group, x2 (for categorical variables) and independent
samples t-tests (for continuous variables) were conducted. To
determine whether infant behavioral response and vagal tone
varied in relation to exposure to FFSF and across episodes of
the FFSF paradigm, three separate ANOVAs were performed
with negative emotionality and RSA ratio index as the
dependent variables: (1) to compare infants at their first
exposure to FFSF, Vagal tone categorization (suppressors vs.
non-suppressors) and Exposure (T1-Exp vs. T2-Ctrl) were
entered as between-subjects variables and Episode (Play,

Vagal Tone and Early Memory for Social Stress

Developmental Psychobiology

Infant Behavioral Response. Negative emotionality

gradually increased across the episodes of the FFSF
(all Fs  8.26, ps  .001; see Figure 1 for means and
standard errors, before the arcsine transformation).
Overall, these findings suggested that negative emotionality increased from Play to Still-Face, all ts  4.29,
ps  .001, indicating the typical still-face effect. Negative emotionality during Reunion was still higher than
during the Play episode, all ts  4.77, ps  .001,
whereas no significant difference was found between
the Still-Face and Reunion episode, suggesting the
typical carry-over effect (Mesman et al., 2009). No
other main or interaction effects emerged for each of
the planned ANOVAs, indicating that no differences in
infants negative emotionality was found in relation to
Vagal tone categorization and exposure to FFSF.

Still-Face, Reunion) as within-subjects factor; (2) to compare

infants with prior exposure to the FFSF and age-matched
infants in the control group, Vagal tone categorization
(suppressors vs. non-suppressors) and Exposure (T2-Exp vs.
T2-Ctrl) were entered as between-subjects variables and
Episode (Play, Still-Face, Reunion) as within-subjects factor;
and (3) to compare infants with the first and second exposure
to FFSF, Vagal tone categorization (suppressors vs. nonsuppressors) was entered as the between-subjects factor,
Exposure (T1-Exp vs. T2-Exp) and Episode (Play, Still-Face,
Reunion) as within-subjects factors. Pairwise comparisons
and t-tests were conducted to further investigate significant
effects. Given the exploratory nature of the present study,
corrections for multiple tests were not applied to avoid risk
for Type II errors (Bender & Lange, 2001; Perneger, 1998).
At the same time, effect sizes are reported to provide a
magnitude measure for significant effects. Effect size was
evaluated by using the partial Eta square (h2p ). The conventional cut-offs for h2p are .01, .06, and .14 for small, medium,
and large effect sizes (Green & Salkind, 2003). Pearson
correlations were used to evaluate the relation between
negative emotionality in the FFSF episodes and RSA
ratio index. The correlations were calculated separately by
Exposure and Vagal tone categorization. All analyses were
performed at a significance level p < .05, using SPSS for
Windows (version 17, Chicago, IL).

Vagal Tone.
Comparisons relative to the first exposure to FFSF.
Figure 2 shows RSA ratio index for suppressors and
non-suppressors for each exposure. No main effect for
Exposure was found in RSA ratio index between
experimental and control group in their first exposure
to FFSF, indicating that overall infants had a similar
vagal reactivity in the two conditions. Furthermore, no
effect emerged for RSA ratio index across FFSF
episodes. A main effect emerged for vagal tone
categorization, F(1,72) 4.68, p .034, h2p .06. This
effect was qualified by interactions between FFSF
episodes and vagal tone categorization, F(2,144)
21.89, p < .001, h2p .23, which indicated that RSA
ratio index differed for both suppressors and nonsuppressors across FFSF. As expected, given the RSA
categorization, follow-up paired t-tests highlighted that
during Play, suppressors showed a higher vagal tone

RESULTS
Infant and Maternal Characteristics
No differences were found between experimental and
control group for infant and maternal characteristics
(all ps > .05; see Table 1 for descriptive statistics).
Distribution of vagal tone categorization did not differ
between experimental and control group (x2 .51,
p > .05).

Table 1. Infants and Mothers Characteristics for Experimental and Control Groups
Experimental Group

Control Group

Vagal Tone Categorization

All
Mean

Suppressors
SD

Mean

SD

Socio-demographic variables
Infant characteristics
Gestational age (weeks) 39.36
1.33
39.39
1.29
Birth weight (grams)
3242.92 361.05 3251.67 383.21
Mother characteristics
Age at the first FFSF
33.75
3.47
32.71
2.55
session (years)
Educational level
13.25
3.16
13.11
3.29
(years of study)
Socio-economic status
48.06
24.71
47.78
25.33

Vagal Tone Categorization

Non-Suppressors

All

Mean

SD

39.33
3234.17

1.41
348.38

34.73

3.99

33.78

4.33

33.39

13.39

3.11

14.13

2.94

48.33

24.79

51.22

25.22

Socio-economic status was coded according to Hollingshead (1975) classification.

Mean

Suppressors
SD

Mean

SD

Non-Suppressors
Mean

SD

39.36
3382.50

1.26
405.03

4.58

34.05

4.22

14.71

4.55

14.37

3.65

58.82

19.65

45.83

27.65

39.34
1.12
39.31
0.95
3296.10 363.63 3174.12 260.27

Montirosso et al.

Developmental Psychobiology

FIGURE 1 Means and standard errors of negative emotionality across the FFSF paradigm for
suppressor (A) and non-suppressor (B) infants in the experimental group (T1-Exp: first exposure,
4 months of age; T2-Exp: second exposure, 4 months 15 days) and control group (T2-Ctrl:
unique exposure, 4 months 15 days). Note. FFSF, face-to-face still-face procedure; raw
measures of negative engagement before arcsine transformation are reported for easy interpretation.

than non-suppressors, M(suppressors) .34, SD .88;

M(non-suppressors) .06; SD .51), t(76) 2.50,
p .023, while a reverse trend emerged in the StillFace episode, in which non-suppressors exhibited a
higher vagal tone than suppressors, M(suppressors)
.39, SD .83; M(non-suppressors) .69, SD .80, t
(76) 5.84, p < .001.

Comparisons between infants with prior exposure to

the FFSF and age-matched infants in the control
group. ANOVA revealed a significant threefold interaction of Exposure  FFSF episodes  vagal tone categorization,
F(2,146) 5.12,
p .007,
h2p .07,
indicating that the RSA ratio index differed across
FFSF for both suppressors and non-suppressors be-

FIGURE 2 Means and standard errors of vagal tone activity (RSA ratio index) across the FFSF
paradigm for suppressor (A) and non-suppressor (B) infants in the experimental group (T1-Exp:
first exposure, 4 months of age; T2-Exp: second exposure, 4 months 15 days) and control group
(T2-Ctrl: unique exposure, 4 months 15 days). Note. FFSF, face-to-face still-face procedure;
RSA ratio index is the natural logarithm of the ratio between RSA in a given FFSF episode and
the baseline RSA value before FFSF get started.

Developmental Psychobiology

tween T2-Exp and T2-Ctrl. This effect was further

specified by the interaction between Exposure and
FFSF episodes, F(2,146) 4.46, p .009, h2p .06 and
by the significant vagal tone categorization  FFSF
episodes interaction, F(2,146) 13.78, p < .001,
h2p .16. Follow-up unpaired t-tests indicated that
during the Still-Face episode suppressors of T2-Ctrl
showed lower RSA ratio index than those of the T2Exp, t(33) 2.22, p .020, whereas no differences
emerged for non-suppressors and for Play and Reunion.
Thus, the vagal tone response was different if infants
were previously exposed or not to maternal unresponsiveness, suggesting an experience effect, even if only
for suppressors.
Comparisons between the first and second exposure
to FFSF. ANOVA revealed a significant threefold
interaction of Exposure  FFSF episodes  vagal tone
categorization, F(2,66) 3.34, p .042, h2p .10, indicating that the RSA ratio index differed between T1Exp and T2-Exp across FFSF and differently for
suppressors and non-suppressors. This interaction was
further evaluated using a two-way ANOVA with vagal
tone categorization as the between-subjects factor and
Exposure (T1-Exp vs. T2-Exp) as a within-subject
factor, separately for each FFSF episode. An interaction
emerged for the Still-Face episode, F(1,35) 4.11,
p .048, h2p .11, but not for Play and Reunion,
suggesting that during Still-Face the suppressors exhibited lower RSA ratio index in T1-Exp (M .37,
SD .85) than in T2-Exp (M .14, SD .80) sessions,
t(17) 2.18, p .044 (see Fig. 2).

Relation Between Infant Behavioral Response

and Vagal Tone
There were no significant correlations between negative
emotionality and RSA ratio index, separately for
suppressors and non-suppressors, suggesting a lack of
correspondence between behavioral and physiologic
reactivity measures during FFSF.

DISCUSSION
The main goal of the study was to examine whether 4month-old infants have a memory for a social stressful
event (i.e., maternal still-face) after a 15-day retention
interval when they were again exposed to the same
social event using RSA and behavior as a measures of
memory. The RSA data partially support the expectation that infants have long-term memory for a social
stressor over the retention interval. When infants in the
experimental group were re-exposed after 2 weeks to
maternal unresponsiveness, the changes in vagal tone

Vagal Tone and Early Memory for Social Stress

response were mediated by individual differences in

physiological reactivity to the first exposure (i.e.,
suppressors vs. non-suppressors). Specifically, infants
who showed RSA suppression during the Still-Face
episode in the first exposure, showed no RSA suppression when re-exposed 2 weeks later to the maternal
still-face. On the other hand, infants who showed no
suppression during the first exposure, showed no
change in RSA during the second exposure to maternal
still-face. Moreover, regardless of individual differences
in vagal categorization, age-matched infants with no
prior exposure to the maternal still-face showed similar
RSA response to that of the experimental infants at
their first exposure. No differences in behavior were
found related to exposure or individual differences.
Overall, findings are consistent with previous study
investigating infants memory for social stress using
salivary cortisol response as a measure of memory
(Montirosso et al., 2013). Nonetheless, the present
work on RSA evidences slightly different patterns for
memory in relation to individual differences. In the
cortisol study, infants who exhibited either an increase
or a decrease in salivary cortisol in response to social
stress showed significant changes in HPA reactivity
when re-exposed to the FFSF 15 days later. In the
present study, non-suppressors showed no change in
RSA from the first to the second exposure to maternal
still-face. At a general level, these findings indirectly
corroborate the idea that stress reactivity is not a
unitary process and that different stress reactivity
systems may only be partially linked during early
infancy (Feldman et al., 2010; Haley & Stansbury,
2003). Moreover, this lack of relationship is suggestive
of a different way through which the neuroendocrine
(i.e., cortisol) and the parasympathetic (i.e., RSA)
systems for stress reactivity affect infant memory for
social events during the first months of life. An
alternative methodological explanation of this dissociation is that there is a substantial lag between the time
resolution of vagal tone activity and the peak cortisol
response of HPA-axis reactivity: the RSA is a real-time
measure whereas salivary cortisol is a measure of
poststress reactivity.
The change in RSA reactivity for suppressors
suggests that these infants autonomic reactivity
markedly changed with experience. A possible interpretation of the disinhibition of the vagal brake (i.e.,
increased RSA) observed during the second FFSF
exposure is that vagal tone suppression during the first
exposure might have been related to an enhanced
memory for the social stressor (i.e., maternal still-face).
If so, the lack of vagal suppression at the second
exposure could be indicative of infants reduced
physiological regulation related to the expectations

Montirosso et al.

about how their mothers will behave in that specific

context. This interpretation seems consonant with
findings from research in non-social cognition, which
has documented that greater vagal tone suppression
during encoding is related to better memory recall
(Haley et al., 2010b). In sum, given that the suppressor
infants might remember the maternal Still-Face episode
as a stressful event, the RSA change might indicate that
infants response was triggered by recall of a prior
exposure to social stress.
Non-suppressor infants showed no significant change
in vagal tone between the first and the second exposure
to maternal unresponsiveness. A plausible interpretation
is that the memory trace modulated by vagal activity
during the first FFSF exposure was weaker in nonsuppressor infants than in suppressor infants. In other
words, non-suppressor infants who exhibited higher
autonomic activity might not encode the stressful event
as robustly as suppressor infants did. Indeed, though
we cannot establish causal relationships between these
variables, this interpretation is supported by previous
studies, which have reported negative relations between
cardiac arousal and attention (Suess et al., 1994).
Moreover, this explanation is consistent with the view
concerning reactivity, limited resources, and infants
poor regulatory abilities (Calkins & Fox, 2002). Nonsuppressor infants autonomic reactivity is reminiscent
of those of reactive individuals who need to expend
effort in order to modulate their higher physiological
arousal at the expense of attending to other important
internal and external information (Porges, 2011). As a
result, non-suppressors would have poorer attention at
their first exposure, which in turn, during the second
exposure, would also contribute to the lack of recall of
earlier stress exposure. On the other hand, it is not
possible to rule out that non-suppressor infants had no
memory for social stress after 15-days. Rather, they
might remember the previous stressful experience, but
their predisposition to react to a challenging condition
with a lowered parasympathetic regulation and greater
arousal might lead them to respond to second FFSF
exposure as if they were facing it for the first time; that
is, as if it were a new experience. This latter possibility
may be particularly salient as the retrieval context is
itself stressful. Whether in non-suppressor infants less
physiological regulation or lower emotion regulation
affect long-term memory storage will require further
investigation.
Similar to prior FFSF studies (Adamson & Frick,
2003; Montirosso, Riccardi, Molteni, Borgatti, & Reni,
2010; Weinberg & Tronick, 1996) infant negative
emotionality increased over the episodes of the FFSF.
Nonetheless, as expected, there were no significant
behavioral differences between the first and second

Developmental Psychobiology

exposure to FFSF, either for suppressors or nonsuppressors. Consistent with a previous study on infant
social memory (Montirosso et al., 2013), when infants
faced the second exposure, they did not show behavioral signs of a recall of the first experience. However, no
differences emerged in negative affect between the first
and second stress exposure, suggesting little or no
association between behavioral and RSA response.
Though a striking lack of linkage, it should be noted
that the emotional components of infant behavior
during the FFSF paradigm do not always correspond
with the changes in autonomic response. Previous
studies produced mixed findings for the association
between infant behavior and cardiac activity measures.
Bazhenova et al., 2001, for example, used a modified
researcher-infant FFSF paradigm, which included object-mediated contexts (i.e., picture attention and toy
interaction) prior to the Still-Face episode. The findings
highlighted that RSA decreased and negative signaling
increased during the still-face condition, suggesting
that the two domains were inversely linked. In contrast,
Moore and Calkins (2004) found that infants behavioral and physiological responses were uncorrelated during the Play episode, while higher levels of negative
affect were related to larger decreases in heart period
from baseline to still-face. In an innovative study using
a modified FFSF paradigm with an additional still-face
reunion sequence to increase the stressfulness of the
procedure, Haley and Stansbury (2003) found a relationship between negative affect and heart rate in the
second Still-Face episode, but not during the first one.
The study suggested that behavioral and physiological
systems may become more tightly coupled particularly
under conditions of greater and repeated stress. In a
conventional FFSF paradigm study, Weinberg and
Tronick (1996) found that despite the lack of differences in RSA between Play and Reunion episodes,
during the Reunion infants continued to exhibit negative affect and higher levels of positive affect than
during the Play episode. More recently, no significant
correlations emerged between vagal tone and infant
distress during a conventional FFSF procedure (Conradt
& Ablow, 2010). Thus, although the discrepancies
reported may be related to differences in the experimental context or study design, it would seem that
physiological and behavioral measures are only partially coupled during early development (Doussard-Roosevelt, Montgomery, & Porges, 2003; Graziano and
Derefinko, 2013). At the same time, it is possible that
these domains may work in concert only under specific
conditions or sequentially over time, however, further
research is needed to confirm these possibilities.
There are limitations to the present study. First, in
the control condition we evaluated infants vagal tone

Developmental Psychobiology

and memory for the interaction only one time at

4 months and 15 days. A more complete experimental
design would have assessed infants at 4 months of age
in the same context but without the still-face manipulation (i.e., in a continuous face-to-face procedure). This
approach would have insured that control infants
experienced the same situational factors (e.g., coming
at lab, seeing the room and experimenters two times, in
a way similar to the experimental group), but without
being exposed to a prior social stress. Nevertheless, it
is noteworthy that the control group at their first and
only exposure exhibited the same behavioral and vagal
tone levels as did the experimental group at T1,
suggesting that the control group was effective in terms
of providing an internal replication of the findings.
Second, findings are based only on 4 month-old infants,
but memory is likely to be moderated by infant age.
For example, older infants with more mature self- and
other-directed regulatory behaviors might better modulate their levels of stress (Melinder, Forbes, Tronick,
Fikke, & Gredeback, 2010), so that they might not
show any memory for the stressful social event after
2 weeks. A third limitation is that we examined only
one retention interval. Thus, we cannot exclude that
infant memory for the stressful event could be also
moderated by the length of the retention interval.
Accordingly, one could expect to find smaller effect
sizes when infants experience a longer retention interval. Finally, effect sizes in the present work ranged
from .06 to .23, which is small-to-large. Specifically,
effect size for the threefold interaction underlining the
memory effect for the experimental group was .10,
which according to Green and Salkind (2003) indicates
a moderate effect size. Overall, given that the study is
among the first to evaluate infants memory for a
social-emotional stressors, further research is needed to
deal with study design limitations and to provide more
focused insights with regard to the early emergence of
this kind of memory during first months of life.
The present study suggests that RSA suppressor
infants remember a social stress at a surprisingly early
age showing a long-term memory persisting for at least
15 days. Thus, the changes in RSA index observed in
the second exposure to FFSF relate to infants previous
experience of social stress and provide new evidence of
the association between changes in autonomic activity
and long-term memory for a stressful social event.
Furthermore, infants memory observed in this study is
apparent for an acute, clearly episodic, social emotional
event (i.e., maternal still-face) with only one distinctive
cue (the yellow smock), rather than for a reiterated or
chronic event. Although one would expect that chronic
reiterated exposures to stressful events, such as neglect,
parental mood disorders or traumatic events to have

Vagal Tone and Early Memory for Social Stress

more lasting memory effects, it will be important to

take into consideration that the mnemonic effects of
these experiences may be mediated by individual
differences in infants characteristic stress reactivity
(Montirosso et al., 2013). Indeed, consideration of
individual differences in reactivity may aid in our
understanding of the varying effects of stress exposure
during early development. In sum, the current study
expands our understanding of infants memory for a
stress experienced during a social interaction and of the
interplay of autonomic activity and long-term memory
for real-life events in the first months of life.

NOTES
The authors wish to thank the nursing and clinician staff of the
Pediatric Unit of the Sacra Famiglia Hospital of Erba (Como),
Italy. Many thanks to Viola Brenna, Erica Casini and Claudia
Fedeli for their help in data collection and technical assistance
with the Face-to-Face-Still-Face procedure. Finally, special
thanks go to all infants and their mothers participating in this
study. This research was supported by funds from the Italian
Health Ministery (Ricerca Corrente 2007 Sviluppo dei processi
di attenzione e di memoria in neonati sani e nati in condizioni
di rischio evolutivo) and grants from NICHD 5R01HD050459
(E.T., PI) and NSF 0819839 (E.T., PI).

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