Physiological and behavioral adaptation to relocation
stress in differentially reared rhesus monkeys: Hair
cortisol as a biomarker for anxiety-related responses
Amanda M. Dettmera,*, Melinda A. Novakb, Stephen J. Suomic,
Jerrold S. Meyerb
aDepartment of Psychiatry, University of Pittsburgh, 3811 O’Hara Street, Pittsburgh, PA, 15213, United States
bDepartment of Psychology, University of Massachusetts Amherst, Amherst, MA, United States
cLaboratory of Comparative Ethology, Eunice Kennedy Shriver National Institute of Child Health & Human Development, NIH,
Bethesda, MD, United States
Received 6 August 2010; received in revised form 19 April 2011; accepted 1 June 2011
Psychoneuroendocrinology (2012) 37, 191—199
both human and nonhuman primates, and hair cortisol is now gaining attention as a biomarker for
stress-related health problems. The present study examined the behavioral and physiological
reactions of rhesus monkey (Macaca mulatta) infants reared in three different rearing environ-
ments to the major stressor of relocation. Infant monkeys (n = 61) were studied from birth
through 2 years of age. For the first 8 months of life, infants were either with their mothers and
peers (MPR, n = 21) or reared in a nursery using either peer-rearing (PR, n = 20) or surrogate-peer-
rearing (SPR, n = 20). At approximately 8 months of age, infants were removed from their rearing
group, simultaneously placed into a large social environment consisting of infants from all three
rearing conditions, and observed for the next 16 months. Behavior was recorded twice per week
from 1 to 24 months, and composite anxiety scores were calculated for each monkey. Monkeys
were initially shaved at the nape of the neck on day 14 to remove any prenatal effects on hair
cortisol deposition. Hair samples were then collected by re-shaving at 6, 12, 18 and 24 months and
analyzed for cortisol content. MPR monkeys were the least affected by the stressor, showing
smaller increases in anxious behavior than the other groups and more rapid physiological
adaptation as assessed using hair cortisol. PR monkeys showed heightened and prolonged anxious
behavior, had the highest cortisol levels prior to relocation, and their cortisol levels did not
decline until more than a year later. SPR monkeys exhibited more rapid behavioral adaptation
than PR monkeys, showing heightened but not prolonged anxious behavior. However, the SPR
group showed a marked increase in cortisol in response to the relocation, and like the PR group,
their physiological adaptation was slower than that of the MPR group as indicated by elevated
Increased hair cortisol concentrations have been associated with stress exposure in
* Corresponding author. Tel.: +1 412 648 1266; fax: +1 412 648 1465.
E-mail address: email@example.com (A.M. Dettmer).
a va ila ble at ww w. scie nce dir ect. com
j our na l h omepa g e: www.e lse vie r.c om/l oca te/ psyne ue n
0306-4530/$ — see front matter # 2011 Elsevier Ltd. All rights reserved.
Exposure to early life adversity has been identified in both
animal and human models as a risk factor for developing
anxiety later in life (Coplan et al., 1996; Huot et al., 2001;
Heim and Nemeroff, 2001). In nonhuman primates, these
effects have been studied in infant monkeys exposed to
different early rearing environments (see review by Stevens
et al., 2009). Infants reared with other naı ¨ve infants for the
first 8 months of life (peer-rearing) develop most species-
typical patterns of behavior but show more intense reactions
to social separation (Higley et al., 1991) and also display
heightened anxiety, particularly in response to novel events,
compared to mother-peer-reared (MPR) monkeys (Harlow,
1969; Suomi, 1991). As juveniles, peer-reared (PR) monkeys
show lower levels of affiliative behavior and are less likely to
have their stress levels reduced by the presence of a com-
panion than normally reared monkeys (Winslow et al., 2003).
Juvenile PR monkeys also show a heightened acoustic startle
response (Parr et al., 2002), enhanced fear potentiated
startle (Nelson et al., 2009), and disrupted sleep patterns
(Barrett et al., 2009) compared to mother-reared controls.
Very little information is available on PR adults. However, in
one study, adult PR animals showed heightened vulnerability
to alcohol consumption compared with normally reared
adults (Fahlke et al., 2000), a finding consistent with the
view that increased anxiety may persist into adulthood fol-
Despite the link between peer-rearing and anxiety, no
consistent pattern has emerged from an examination of
hypothalamic—pituitary—adrenocortical (HPA) axis function
in these infants. PR monkeys have been reported to show
higher basal levels of cortisol (Barrett et al., 2009; Higley
et al., 1992), lower basal levels but no difference in stress
responsivity (Shannon et al., 1998), no difference in basal
levels but lower stress responsivity (Clarke, 1993), higher
stress responsivity (Fahlke et al., 2000), a lower cortisol set
point (Capitanio et al., 2005), and lastly no effects in either
baseline or stress responsivity (Winslow et al., 2003). Varia-
tion in rearing practices or other differences between pri-
HPA function has also been examined in a second kind of
early nursery rearing environment: surrogate-peer-rearing
(SPR). In this condition, infants are reared with inanimate
surrogate mothers and given up to 2 h of daily play experi-
ence with other infants. The purpose of this rearing condition
is to reduce the profound attachment that occurs between
infants, thereby presumably reducing anxiety and facilitating
play behavior. Although less is known about the effects of this
rearing procedure, infant monkeys appear to acquire all the
typical forms of species-normative behavior without showing
excessive clinging (Hansen, 1966). As adults, SPR monkeys
are indistinguishable from mother-reared monkeys in their
reproductive and parental behavior (Novak et al., 1992;
Sackett et al., 2002). Converging lines of evidence suggest
that infant SPR monkeys have significantly lower basal con-
centrations of circulating cortisol (Capitanio et al., 2005)
than MPR and PR counterparts, although in one study (Daven-
port et al., 2003), this difference was present only in the first
month of life. Other studies have revealed that SPR monkeys
respond significantly less to the stress of brief social separa-
tion (Meyer et al., 1975; Shannon et al., 1998) than both MPR
monkeys and PR monkeys, which is perhaps not surprising
given the inanimate nature of their mother and their
restricted interaction with peers.
One of the limitations of previous research on HPA activity
in differently reared monkeys is a reliance on blood plasma as
the primary sample matrix, given the well-known lability of
circulating cortisol to circadian variation and environmental
disturbances. Moreover, studies of HPA function over the
course of development necessitate repeated sample collec-
tions, each requiring capture and sedation of the animals,
which not only further stresses the subjects but may also
confound the results. A long-term measure of circulating
glucocorticoids would greatly reduce the frequency of sam-
pling required and would eliminate the confounds imposed by
capture and restraint.
As a potential alternative to measuring cortisol ‘‘point
samples’’ in plasma or saliva, hair cortisol has recently gained
attention as an index of chronic HPA activity (Davenport
et al., 2006; Sauve ´ et al., 2007; Kirschbaum et al., 2009)
and as a possible biomarker for stress-related health disor-
ders in both human and nonhuman primate populations. Hair
cortisol has proven to be a reliable indicator of the effects of
various stressors. In adult rhesus monkeys, relocation to a
different environment resulted in elevated hair cortisol along
with marked behavioral changes and sleep disturbances
(Davenport et al., 2008). These hair cortisol samples were
also strongly correlated with salivary cortisol samples
obtained in the same time frame (Davenport et al., 2006).
In human studies, increased hair cortisol concentrations have
been associated with perceived stress in healthy pregnant
women (Kalra et al., 2007), hospitalization of at-term infants
cortisol levels at 18 months. By 24 months of age (16 months after relocation), all rearing groups were
indistinguishable from one another physiologically and behaviorally. Spearman rank correlation
revealed that hair cortisol taken at month 6 was not correlated with composite anxiety scores from
months 6 to 8 (just before the relocation), but was positively correlated with composite anxiety
scores between months 8 and 12 (immediately after relocation) for PR infants only (rs= 0.75,
p < 0.001). Month 6-hair cortisol tended to positively correlate with composite anxiety scores for the
following 6 months (months 12—18) for PR monkeys only (rs= 0.47, p = 0.037), which exhibited more
anxious behavior than MPR and SPR infants during this period (ANOVA: F(2,60)= 14.761, p < 0.001)
This is the first study to show that elevated hair cortisol early in life is a biomarker for the later
development of anxious behavior in response to a major life stressor, particularly for infant monkeys
exposed to early life adversity in the form of peer-rearing.
# 2011 Elsevier Ltd. All rights reserved.
A.M. Dettmer et al.
(Yamada et al., 2007), periods of elevated cortisol secretion
in patients with Cushing’s syndrome (Thomson et al., 2010),
and long-term unemployment (Dettenborn et al., 2010).
The present study was designed to determine how differ-
ences in early experience affect the response of infant rhesus
monkeys to the major stress event of relocation by measuring
both anxious behavior and chronic concentrations of cortisol
derived from hair. Repeated assessment of both variables
over 2 years enabled us to test the hypothesis that heigh-
tened HPA activity (as measured by hair cortisol concentra-
tions) early in development predicts later anxious behavior in
response to stress. Another novel and important aspect of
this study was examining anxious behavior and HPA axis
activity in two different nursery-rearing environments (PR
vs. SPR) in comparison to MPR controls. In this regard, the
rhesus monkey infants used this study were part of a larger
project aimed at understanding biobehavioral development
at the Laboratory of Comparative Ethology at the National
Institute of Child Health and Human Development. Infants
were reared in all three rearing conditions described above
for the first 8 months of life and then, as is standard practice
at this laboratory, were relocated and placed together in one
large social group. That this relocation can be considered
initially stressful is based not only on previous findings of
substantial relocation effects in adult rhesus monkeys
(Davenport et al., 2008) but also on the facts that the new
environment was substantially different from the monkeys’
previous housing environments, both in size and complexity,
and in sheer number of animals with which to interact.
Thus, the aims of the present study were (1) to character-
ize hair cortisol concentrations in differently reared infant
rhesus monkeys across the first 2 years of life both before and
after the stressor of relocation and (2) determine whether
hair cortisol was a predictor of anxious behavior after the
major social stressor of weaning and relocation in these
2.1. Subjects and rearing
The subjects were 61 infant rhesus monkeys born and raised
at the Laboratory for Comparative Ethology (LCE) at the
National Institutes of Health in two birth cohorts (2006,
n = 25; 2007, n = 36; 33 males, 28 females). Animals from
three different rearing conditions were studied: mother-
peer-reared (MPR, n = 21), peer-reared (PR, n = 20), or sur-
rogate-peer-reared (SPR, n = 20; Table 1). Rearing conditions
in this laboratory have previously been described in detail
(Ruppenthal, 1979; Shannon et al., 1998). Briefly, MPR infants
were born and reared in harem groups consisting of 6—8 adult
females, 1—2 adult males, and several same-aged infants. PR
and SPR infants were separated from their mothers within the
first 3 days of life and reared in a nursery where they were
housed in incubators and hand-fed for the first 14 days of life.
From days 15 to 37, these infants were placed alone in a
nursery cage with an inanimate cloth-covered surrogate, and
on day 37 PR infants were permanently placed in a large cage
with three other agemates while SPR infants began daily 2 h
play sessions with three other peers. These monkeys were
part of a larger, long-term longitudinal research program at
the LCE to examine the effects of early rearing environments
on socio-emotional development.
2.2. Major life stress event
Infants remained in the MPR, PR, or SPR condition for the first
8 months of life, at which time all infants in a cohort (birth
year 2006 or 2007) representing each of the three rearing
conditions were removed from their housing situations and
placed together into a single social group comprised of
approximately 60 animals. MPR infants were weaned from
their mothers while PR and SPR infants were weaned from
their surrogates. All subjects were housed together in one
large social group and were moved back and forth between
two different housing environments for husbandry purposes
(i.e., two connected indoor enclosures vs. a combined
indoor/outdoor enclosure). The indoor enclosures each mea-
sured 7.3 m ? 3.4 m ? 3.7 m and were equipped with
perches, barrels, swings, and wood shavings. The outdoor
enclosure was a circular corn-crib enclosure measuring
5.03 m in diameter by 5.49 m high. All subjects had free
access between the indoor and outdoor housing areas, except
when they were partitioned to either side for routine clean-
ing or for social behavior observations, or to the inside during
inclement weather (e.g., 4 8C or below). Water was provided
ad libitum and monkeys were fed Purina Monkey Chow
(#5038) and provided enrichment daily. Relocation occurred
for all infants in a cohort on the same day and represented a
major social challenge in the lives of the young monkeys, as
they had to adapt to a new environment and new peers,
including the establishment of a new social hierarchy. This
major life stress served as a valuable opportunity to study
anxious behaviors and physiological responses following such
2.3. Hair cortisol measurements
Hair was shaved from each infant at the back of the neck
during a routine neonatal assessment on day 14. Shaving at
this time allowed for adequate re-growth of hair for cortisol
analysis over the next 5.5 months as well as a precise window
of time to which the accumulated cortisol would correspond.
Additionally, removing the hair at this age insured that
prenatal sources of cortisol (both fetal and maternal) would
be removed from the sample (Dettmer et al., 2009). Hair was
allowed to re-grow and shaved again during routine health
procedures at months 6, 12, 18, and 24. For these very young
monkeys, a 6-month delay between sample collections was
necessary to ensure that an adequate amount of hair was
present for cortisol analysis (?250 mg; see Davenport et al.,
2006). The samples at 6, 12, 18, and 24 months were analyzed
Subject characteristics for this study.
Hair cortisol predicts anxiety in young rhesus monkeys
by means of a sensitive and specific enzyme immunoassay kit
(#1—3002; Salimetrics, State College, PA) for cortisol content
according to procedures developed and validated by our
laboratory (Davenport et al., 2006, 2008). Briefly, hair was
washed twice with isopropanol and allowed to dry for 5—7
days before being ground to a fine powder. Approximately
50 mg of the powder was incubated overnight in 1 mL of
methanol to extract the cortisol, then centrifuged, and the
supernatant transferred to a new tube to be dried down
under nitrogen gas. The cortisol extract was reconstituted
with 400 mL assay buffer and analyzed according to the
manufacturer’s recommendations. Hair cortisol samples
were assayed after each 6-month period, and samples from
all three rearing conditions were approximately balanced
across each assay plate. Inter- and intra-assay coefficients of
variation were <10%.
2.4. Anxiety scores
From day 37, when the PR and SPR infants were placed into
social groups, through 2 years of age, each infant was
observed for 5 min twice per week (once in the morning
and once in the afternoon) using focal animal sampling.
The order of infants to be observed was randomized across
morning and afternoon sessions, and from months 2 to 8 all
infants were observed in their respective social situations
(i.e., harem group housing for MPR infants, nursery contin-
uous group housing for PR infants, and nursery 2 h daily play
sessions for SPR infants). From months 8 to 24 all infants were
observed in the large mixed-sex housing condition described
above. Durations and frequencies of exploratory, social, and
solitary behaviors were recorded using JWatcher software
(Blumstein et al., 2006). Based on previous research describ-
ing anxious behavior in rhesus monkeys (Maestripieri et al.,
1991; Suomi, 1997; Erickson et al., 2005; Karere et al., 2009),
we focused particularly on clinging, fear vocalizations, hud-
dling, self-rocking, self-clasping, self-biting, and scratching.
For each observational session, a composite anxiety score
was calculated by summing the total duration of these
behaviors as many of these behaviors commonly occur with
low frequency (Maestripieri et al., 1991; Erickson et al.,
2005; Karere et al., 2009). To relate the hair cortisol mea-
surements to behavior, mean durations of anxiety were first
calculated for the following blocks: months 2—6, 6—8, 8—12,
12—18 and 18—24. The 6—8 and 8—12 month blocks were
calculated separately to reflect the time periods before and
after housing relocation occurred at 8 months of age. The
hair cortisol concentrations at each 6-month interval were
correlated with the corresponding anxiety blocks (i.e.,
month 6-hair with anxiety from months 2 to 6; month 12
hair for anxiety from months 6 to 8 and 8 to 12; etc.). Due to
the low frequency of the individual behaviors, they were not
separately correlated with hair cortisol.
2.5. Statistical analysis
Hair cortisol and composite anxiety scores at each 6-month
interval were examined for normal distribution using the
Shapiro—Wilk normality test. Hair cortisol at months 6, 18,
and 24, as well as anxiety at months 6, 12, 18, and 24, were
all right-skewed and Tukey’s ladder of transformations was
employed to identify the best transformation for the data
prior to using the variable in any analyses (Tukey, 1977).
Mixed-design ANOVAs were used to analyze the change in
both hair cortisol and composite anxiety scores across the
first 2 years of life, with age (6, 12, 18, and 24 months) as the
within-subjects factor and sex and rearing condition (MPR,
PR, and SPR) as the between-subjects factors. If the assump-
tions of sphericity were violated in any of these analyses, the
Greenhouse—Geisser correction was employed.
Spearman rank correlations were used to determine
whether individuals with higher hair cortisol values at month
6 also exhibited more anxious behavior at 6, 12, 18, and 24
months. These correlations were used to further examine this
relationship from 6 to 8 months of age (immediately pre-
relocation) and from 8 to 12 months of age (immediately
post-relocation). Correlations were performed first for all
subjects together and then for each rearing group separately.
Between months 18 and 24, 9 MPR and 8 PR infants were
unexpectedly removed from the social group for reassign-
ment to another protocol. Thus, hair cortisol data were
available for all 61 infants up through month 18, but for only
44 of the infants at month 24. As this transfer of animals
occurred at approximately 20 months of age, some beha-
vioral data were available for all subjects during the 18—24
month period. Thus the repeated measures ANOVA for hair
cortisol were performed on the 44 subjects for which a hair
cortisol sample was available at each time point.
SPSS software was used for all analyses. An a < 0.05 was
considered statistically significant for the ANOVAs, whereas
we adjusted the alpha level for rejection to 0.005 to adjust
for the number of comparisons for the correlational analyses.
3.1. Hair cortisol concentrations across
Repeated measures ANOVA revealed a significant within-
subjects effect of age on hair cortisol concentrations across
the first 2 years of life (F(3,123)= 24.25, p < 0.001), reflecting
an overall decrease with age (Fig. 1a). When specific time
points were analyzed using paired sample t-tests, overall hair
cortisol concentrations (i.e., collapsed across rearing condi-
tion) significantly decreased between months 12 and 18
(t(58)= ?4.52, p < 0.001) and again between months 18
and 24 (t(44)= ?4.43, p < 0.001). A significant age ? rearing
was also observed (F(6,123)= 4.95,
p < 0.001; Fig. 1b), indicating different patterns of hair
cortisol levels across time in the various rearing groups.
When we compared the groups with each other at each time
point using simple effects ANOVAs, the PR infants exhibited
the highest hair cortisol concentrations at month 6
(F(2,59)= 4.78, p = 0.01), and both PR and SPR infants exhib-
ited higher concentrations than MPR infants at month 18
(F(2,60)= 12.56, p < 0.001). Interestingly, by month 24 the
rearing groups were indistinguishable from one another.
When changes over time were analyzed according to rearing
condition, we found a significant rise in hair cortisol values
for SPR infants from months 6 to 12 (during the period
involving the relocation; t(19)= ?3.30, p = 0.004). Significant
decreases in hair cortisol were observed in the MPR infants
A.M. Dettmer et al.
between months 12 and 18 (t(19)= ?5.84, p < 0.001), and in
p = 0.002)
p = 0.001) infants between months 18 and 24. No main effects
of rearing or sex were observed, nor any interaction between
these two variables.
3.2. Anxious behavior across development
Repeated measures ANOVA (Greenhouse—Geisser estimate)
revealed a significant within-subjects effect of age on anxi-
(F(2.62,146.42)= 116.52, p < 0.001). Composite anxiety scores
sharply increased between months 6 and 12, soon after the
major life stressor of housing relocation (t(58)= ?11.53,
p < 0.001), stayed elevated between months 12 and 18,
and declined to below pre-stress levels by 24 months of
age (t(60)= ?4.20, p < 0.001; Fig. 2a). A main effect of
rearing was observed (F(2,56)= 5.44, p = 0.007) such that
overall, PR infants exhibited the most anxious behavior,
followed by SPR then MPR infants (data not shown). Simple
effects ANOVA at each time point revealed that anxious
behavior remained elevated between months 12 and 18 in
the PR group (F(2,60)= 13.77, p < 0.001; Fig. 2b), whereas
MPR and SPR infants did not differ at this age. By 24 months of
age, the rearing groups were indistinguishable from one
another. No other interactions were revealed.
3.3. Hair cortisol and anxiety score relationships
Spearman correlations revealed a significant positive corre-
lation between month 6-hair cortisol and anxious behavior
from months 6 to 12 for all subjects combined (rs= 0.53,
**Differs from every other time point ( p < 0.001). (b) Rearing differences in anxious behavior across the first 2 years of life.
**PR > MPR = SPR ( p ? 0.01). Data shown as mean ? SEM.
(a) Anxious behavior rose and remained elevated after the major life stressor at month 8, then returned to pre-move levels.
24 < months 6, 12, and 18 ( p < 0.01). (b) Rearing differences in hair cortisol across the first 2 years of life. *PR > MPR = SPR ( p < 0.05);
**PR = SPR > MPR ( p < 0.01). Data shown as mean ? SEM.
(a) Hair cortisol concentrations declined across the first 2 years of life. *Month 18 < month 12 ( p < 0.05); **month
Hair cortisol predicts anxiety in young rhesus monkeys
p < 0.001). When each rearing group was examined sepa-
rately, only individuals from the PR group exhibited signifi-
cant positive correlations between month 6-hair cortisol and
anxious behavior between months 6 and 12 (rs= 0.71,
p = 0.001). To further determine whether this association
held for the time period immediately before relocation
(i.e., 6—8 months of age) or immediately after relocation
(i.e., 8—12 months of age), the data were analyzed sepa-
rately at each of these time points. Month 6-hair cortisol was
not significantly correlated with anxious behavior between
months 6 and 8 for any rearing group, but was strongly
correlated with anxious behavior from months 8 to 12 for
all infants combined (rs= 0.41, p < 0.001; Table 2). Once
again, when each rearing group was examined separately,
only the PR infants exhibited this relationship (rs= 0.75,
p < 0.001; Table 2). PR infants tended to maintain a positive
correlation between month 6-hair cortisol and anxiety-like
behavior for the subsequent 6 months (i.e., from month 12 to
18; rs= 0.47, p = 0.037). Post hoc regression analysis revealed
that month 6-hair cortisol was indeed a significant predictor
of anxious behavior from months 12 to 18 for PR infants
(R2= 0.24; p = 0.03), but not for MPR (R2= 0.02; p = 0.58) or
SPR (R2= 0.08; p = 0.23) infants.
The present study is the first to observe differently reared
monkeys from birth continuously through the first 2 years of
life and the first to use a chronic measure of HPA axis activity
to characterize changes in this system in response to the
stress of relocation. Our study demonstrated that hair corti-
sol is a potential biomarker for the development of anxiety-
like behavior in response to a major life stressor in young
monkeys, particularly in animals exposed to adverse early
rearing. It also revealed significant divergence in the
responses of the two nursery rearing groups (PR and SPR)
to relocation in comparison to MPR controls.
A major finding of the present study is that PR infants with
higher hair cortisol levels measured early in life before
relocation exhibited more anxious behaviors in the months
immediately following relocation, a relationship that tended
to persist for the subsequent 6 months. The PR condition is a
well-established model of anxiety in humans (Suomi, 1981;
Higley and Suomi, 1989; Higley et al., 1990; Fahlke et al.,
2000), and our findings are consistent with both the human
and animal literature in which individuals exposed to adverse
conditions early in life are more prone to exhibiting anxiety
after a subsequent major stressor (Suomi, 1997; Francis
et al., 1999; Ladd et al., 2000; Heim and Nemeroff, 2001).
However, the present results are the first to demonstrate a
prolonged reaction in PR infants over many months and are
also unique in showing that a biological measure obtained
early in life significantly predicts later stress-related anxious
behavior in these same subjects.
Another major finding is the diversity of response to
relocation in the two nursery rearing groups both in terms
of anxious behavior and HPA axis activity. Consistent with
previous studies, we found that PR infants exhibited more
anxious behavior than MPR infants following exposure to a
stressor (Barrett et al., 2009; Higley et al., 1991); however,
this is the first study to show that SPR infants exhibited less
anxious behavior than their PR counterparts in reaction to the
same stressor. Our findings lend support to previous results
indicating that SPR infants are behaviorally more similar to
MPR than are PR infants (Sackett, 1982; Ruppenthal et al.,
1991; Strand and Novak, 2005). Indeed, MPR and SPR infants
showed more rapid behavioral adaptation to the relocation
stressor as evidenced by their reduced anxiety scores in the
12—18-month period, whereas PR infants showed elevated
anxiety scores across two consecutive 6-month periods
(months 6—12 and 12—18). One possible explanation for this
finding is that like MPR infants, SPR infants develop an
attachment bond with their inanimate mother (i.e., surro-
gate) and develop playful relationships with their peers,
whereas PR infants lack this distinction. PR infants must
serve as both attachment figures and playmates, making it
difficult for the monkeys to dissociate the two roles.
A somewhat different pattern of results was obtained for
HPA axis activity. Our finding revealed that PR infants exhib-
ited the highest hair cortisol at 6 months of age prior to
relocation is in agreement with previous studies describing
elevated ‘‘point’’ measures of cortisol (e.g., in CSF or blood
plasma) in these infants (Barrett et al., 2009; Higley et al.,
1992). However, only one group, SPR infants, showed a
significant increase in hair cortisol concentrations from 6
to 12 months during the period in which the housing reloca-
tion occurred. Because all groups exhibited increases in
anxious behavior during the same time period, we cannot
attribute this rearing condition effect to an overall lack of
responsiveness of the PR and MPR groups. For the PR group, it
is possible that the absence of a change was due, in part, to
the already high cortisol levels expressed in these monkeys
prior to relocation. The lack of HPA axis reactivity in the MPR
group may be related to the relatively lower anxiety levels
shown by this group in response to relocation. Naturally
occurring age-related declines in basal cortisol values also
This relationship tends to persist for the subsequent 6 months (12—18 months).
Month 6-hair cortisol is correlated with anxious behavior immediately following relocation (8—12 months) for PR infants.
Hair cortisol 6 month
Hair cortisol 6 month
Hair cortisol 6 month
Hair cortisol 6 month
*p < 0.05.
**p < 0.001.
A.M. Dettmer et al.
might have influenced this pattern (Lewis and Ramsay, 1995;
Guazzo et al., 1996). However, this hypothesis would best be
tested by examining hair cortisol from infants reared with
their mothers who did not undergo the relocation stress;
indeed, we are currently collecting and examining such data
on a subset of infants at the LCE. Finally, rearing condition
differences in hair cortisol were also observed in the present
study at month 18, a time at which the MPR infants exhibited
lower hair cortisol concentrations than either PR or SPR
infants. Taken together with the behavioral data, these
findings suggest that although the level of HPA activity prior
to stress onset predicted subsequent anxiety responses to the
stressor, changes in HPA activity after stress onset did not
parallel the changes in anxiety when all of the rearing
conditions are considered.
One of the intriguing outcomes of this study was the
finding that at 24 months of age (16 months after the
relocation), all rearing groups showed similar levels of
anxious behavior and HPA axis activity. The extent to which
this outcome may have resulted from the mixing together of
the different rearing groups (as opposed to maintaining them
with animals only of the same rearing condition) is an impor-
tant question that cannot be answered with the available
data. It is interesting to note that a number of different
primate facilities routinely house young monkeys in mixed
rearing groups for variable periods of time after the first 6—8
months of life. However, despite the present finding of
similar levels of anxiety-like behavior across rearing groups
at 24 months of age, other studies have demonstrated that
the mixing of differently reared monkeys does not eliminate
all behavioral differences later in life. For example, juvenile
PR monkeys with this mixed rearing exposure show sleep
disturbances (Barrett et al., 2009) and enhanced fear-poten-
tiated startle (Nelson et al., 2009) compared to mother-
reared controls. Furthermore, studies at the LCE where
animals typically receive extensive mixed rearing exposure
show heightened vulnerability of PR monkeys to alcohol
consumption in adulthood compared to their MPR counter-
parts (Fahlke et al., 2000). Thus, the effects observed in our
study are best viewed from the perspective of adaptation to
the mixed housing condition following establishment of a new
set of relatively stable social relationships. When additional
stressors or novel situations are encountered, rearing condi-
tion differences may once again be discerned, thus explain-
ing some of the disparate findings mentioned above. In any
case, further research is needed to determine the role that
physical and social contact with same- vs. differently-reared
age mates might play in modulating anxious behavior in young
monkeys. Additionally, further research is warranted to
examine normative age-related changes in hair cortisol con-
centrations in mother-reared monkeys who do not undergo
the stress of relocation, as the current study did not include
such infants. Indeed, we are currently collecting and begin-
ning to analyze hair samples from mother-reared infants who
remain with their mothers for at least the first year of life at
In summary, we have demonstrated that PR, SPR, and MPR
infant rhesus monkeys showed rearing condition-dependent
patterns of behavioral and physiological reactivity to a major
social stressor. All infants exhibited increased anxiety-like
behavior following imposition of the stressor, but subsequent
recovery from the anxiety response as well as the HPA
response measured using hair cortisol differed across the
three groups. Interestingly, early hair cortisol levels were
shown to predict later anxious responses to a stressor in the
at-risk PR monkeys. Finally, the present data also address a
gap that has existed in nonhuman primate research, as the
majority of previous studies have focused on infants in the
first 6 months of life and/or on animals in early adulthood and
beyond. While further studies are needed to resolve some of
the questions raised by the present findings, it seems likely
that measurement of hair cortisol concentrations will prove
increasingly valuable in the search for valid biomarkers of
stress-related and psychiatric disorders.
Role of funding sources
This research was supported by funds from the Division of
Intramural Research, National Institute of Child Health &
Human Development, NIH, and by NIH Grant RR11122 to
M.A.N. Neither the NIH nor the NICHD had any further role
in study design; in the collection, analysis and interpretation
of data; in the writing of the report; and in the decision to
submit the paper for publication.
Conflict of interest
We declare that none of the authors has any financial or other
relationships that might lead to a conflict of interests in
relation to this study or the content of this manuscript.
The studies described in this report were performed in
accordance with the NIH Guide for the Care and Use of
Laboratory Animals and were approved by the NICHD Animal
Care and Use Committee. These findings represent a portion
of AMD’s doctoral dissertation research, and were presented
at the 42nd annual meeting of the International Society for
Developmental Psychobiology in Chicago, IL, October 14—17,
2009. We are grateful to Matthew Novak, Angela Ruggiero,
Elizabeth Mallott, Nicole Bowling, Lisa Darcey, and Daniel
Hipp for assistance with hair cortisol collection. We owe
thanks to Elizabeth Henchey, Brian Kelly, Kaushal Jani, and
Karen Stonemetz for assistance with hair cortisol assays. We
thank Annika Paukner and Nicole Bowling for assistance in
behavioral data collection.
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