Psychiatry Research 210 (2013) 575–583

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Psychiatry Research

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Blunted stress cortisol reactivity and failure to acclimate to familiarstress in depressed and sub-syndromal children

Hideo Suzuki a,n, Andy C. Belden a, Edward Spitznagel b, Rachel Dietrich a, Joan L. Luby a

a Department of Psychiatry, Washington University School of Medicine, St. Louis, MO, USAb Department of Mathematics, Washington University in St. Louis, St. Louis, MO, USA

a r t i c l e i n f o

Article history:Received 1 June 2012Received in revised form20 June 2013Accepted 23 June 2013

Keywords:Hypothalamic–pituitary–adrenal axisSalivary cortisolChildMajor depressive disorderPreschoolHigh-risk

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espondence to: Washington University Schoory, 660 South Euclid Avenue, Campus Box 8134314 747 0001.ail address: suzukih@psychiatry.wustl.edu (H.

a b s t r a c t

Depressed adults have shown blunted or elevated cortisol reactivity in response to various forms ofpsychosocial stress. However, there have been few studies of cortisol reactivity in children who had earlyonset depression or a history of depression during the preschool–school period. The present studyutilized a laboratory stress paradigm and collected salivary cortisol from preschoolers at baseline (ages 3–5 years) and 24-month follow-up (ages 5–7 years). Repeated-measures multivariate analyses of variance(MANOVAs) were used to compare cortisol reactivity to mild stress between children with MajorDepressive Disorder (MDD), elevated symptoms of depression (sub-syndromal MDD), and healthycontrols. For healthy children, a quadratic cortisol reactivity curve was found at baseline (n¼73), whichappeared flatter under similar stressful situations at follow-up (n¼14), which may reflect acclimation tothe paradigm. In contrast, children with MDD (n¼46) and sub-syndromal MDD (n¼76) showed a peakcortisol response to the novelty of lab arrival and then reduced and blunted responses to stressors atbaseline. These cortisol responses persisted at follow-up in children with a history of MDD (n¼41) orsub-syndromal MDD (n¼73). These results suggest that the hypothalamic–pituitary–adrenal (HPA) axisshows a blunted response to stress and failed to acclimate to familiar stressful situations in depressedand sub-syndromal depressed children.

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1. Introduction

Excessive secretion of cortisol (hypercortisolemia) is a well-established biological correlate of Major Depressive Disorder (MDD)(Nestler et al., 2002; Pariante and Lightman, 2008), although itsdiagnostic utility has been minimized by its lack of specificity(Braddock, 1986; Gillespie and Nemeroff, 2005). Depressed indivi-duals (at ages 8–78 years) showed higher cortisol levels throughoutcircadian periods (Lopez-Duran et al., 2009; Stetler and Miller, 2011),at sleep onset (Kaufman et al., 2001), at pre-stress (Gotthardt et al.,1995; Young et al., 2000), and/or at post-stress (Heim et al., 2000b),compared to healthy controls. Alternatively, constant cortisol levelsfollowing stressful situations (blunted cortisol reactivity to stress), asopposed to a dynamic change in cortisol, have been reported inseveral studies of depressed adults, adolescents, and children (Burkeet al., 2005; Lopez-Duran et al., 2009).

However, the issue of whether or not stress cortisol reactivity isaltered in childhood/adolescent MDD has been less straightfor-ward (Kaufman et al., 2001; Burke et al., 2005; Stetler and Miller,2011). Only a few studies have examined cortisol reactivity to

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l of Medicine, Department of, Saint Louis, MO 63110, USA.

Suzuki).

stress in young children (Lopez-Duran et al., 2009). Examining apattern of cortisol reactivity in Preschool Onset MDD (PO-MDD) isparticularly important, given recent findings illustrating long-termand deleterious psychological, biological, and social trajectoriesassociated with PO-MDD (Luby et al., 2009b, 2009c). PO-MDD is apsychiatric disorder characterized by the Diagnostic and StatisticalManual of Mental Disorders, Fourth Edition (DSM-IV) depressivecriteria (except the 2-week duration criterion) that are develop-mentally adjusted for age appropriate manifestations in somecases that occurs during the preschool ages (i.e., under the ageof 6 years). A growing body of empirical data provides validationfor PO-MDD (Luby et al., 2003b, 2006, 2009a, 2009c; Doughertyet al., 2011a), and several epidemiologic studies have detecteddepressed preschoolers (Egger and Angold, 2006; Lavigne et al.,2009; Wichstrom et al., 2012; Bufferd et al., 2012). Luby et al.(2003a, 2004) reported that depressed preschoolers at ages 3–6years showed blunted cortisol reactivity, with elevations in cortisollevels, in contrast to healthy controls who exhibited a dynamiccortisol reactivity curve. Note that, unlike healthy adults, healthypreschoolers (Luby et al., 2003a; Dougherty et al., 2010, 2011b) andadolescents (Rao et al., 2008) showed a quadratic reactivity curvewith two peaks of cortisol levels—upon arrival to the novellaboratory environment and at the end of a laboratory stressparadigm. Nevertheless, this quadratic reactivity curve was dis-rupted in PO-MDD (Luby et al., 2003a, 2004).

H. Suzuki et al. / Psychiatry Research 210 (2013) 575–583576

Cortisol levels among the children of depressed mothers, agroup deemed to be at high risk for MDD, have also been examined.This high-risk group showed high circadian cortisol levels, orheightened cortisol reactivity to stress (Essex et al., 2002; Brennanet al., 2008; Feldman et al., 2009), depending on other psychologicalfactors (Ashman et al., 2002; Dougherty et al., 2011b). MaternalMDD may impact cortisol secretion in offsprings through geneticmechanisms, as suggested by evidence that preschoolers withgenotypes known to confer high risk for MDD (i.e., the short-5-HTTLPR and the Met-BDNF allele carriers) exhibited elevations incortisol under stress (Dougherty et al., 2010).

Therefore, cortisol reactivity appears to be blunted and/orheightened not only in children with MDD but also in those athigh familial/genetic risk for MDD. However, it is unclear whethernon-depressed children who are at a high risk for MDD due totheir expression of sub-clinical symptoms (sub-syndromal MDD)show alterations in cortisol reactivity. In depressed adolescentsand adults, blunted cortisol reactivity is more apparent as MDDseverity increases (Burke et al., 2005; Harkness et al., 2011),suggesting that cortisol reactivity may be altered in sub-syndromal MDD, compared to non-depressed controls. There is agap in literature pertaining to cortisol reactivity in sub-syndromalMDD in childhood.

Furthermore, it also remains unclear how cortisol reactivity inchildren changes as a consequence of acclimation to stressfulsituations. According to the allostatic load model, healthy indivi-duals are expected to optimize a physiological response to adapt tothe demands of the environment (Juster et al., 2010; McEwen andGianaros, 2010). Based on this model, it is possible that healthychildren may initially show a quadratic reactivity curve to newstressors at baseline assessment (as discussed above), but thenshow a diminished its reactivity to identical stressors at follow-upassessment due to acclimation to stressful situations. In contrast,this pattern may depend on depression severity, such thatdepressed children may fail to optimize and adjust their cortisolresponse to the familiar stressful situations (i.e., lack of adaptation)(McEwen, 1998; Juster et al., 2010). To our knowledge, no studieshave comprehensively examined a potential effect of MDD severityon cortisol reactivity at baseline and follow-up assessments. Toclarify this issue, it is necessary to use a similar stress paradigmand assess cortisol reactivity across different waves.

Therefore, the present study aimed to address how stresscortisol reactivity over a 24-month period differed among childrenwith current or past history of MDD, sub-syndromal MDD, andhealthy status. To achieve this goal, cortisol reactivity to similarmild laboratory-induced stress was repeatedly assessed at twoperiods: baseline and 24 months later. We hypothesized thathealthy childrenwould initially show a quadratic cortisol reactivityduring a baseline stress paradigm because of its novelty (Lubyet al., 2003a, 2004; Rao et al., 2008), but would show no cortisolreactivity during a follow-up stress paradigm because they havebecome familiar with it. In contrast, children with MDD and sub-syndromal MDD would show blunted or heightened cortisolreactivity at the baseline (Luby et al., 2003a, 2004; Burke et al.,2005; Lopez-Duran et al., 2009) and would show similar cortisolreactivity at the follow-up because of lack of adaptation (McEwen,1998; Juster et al., 2010).

2. Methods

2.1. Participants

This investigation is a multi-method and multi-informant longitudinal studydesigned to examine the nosology, course, and psychophysiological correlates ofPO-MDD. Children between 3.0 and 5.11 years were recruited from pediatricians'offices, daycares, and preschools in the St. Louis metropolitan area, using the

Preschool Feelings Checklist (PFC) (Luby et al., 1999, 2009c). Preschoolers withdepressive symptoms were oversampled to obtain a large sub-sample of this group.Children with chronic medical illnesses, neurological problems, pervasive devel-opmental disorders, language and/or cognitive delays were excluded. A total ofN¼306 caregiver–child dyads agreed to participate at baseline (T1), N¼271 dyadscompleted the 12-month follow-up assessment (T2), and N¼257 dyads completedthe 24-month assessment (T3); that is, N¼257 children had longitudinal data at T1,T2, and T3. The Institutional Review Board (IRB) at the Washington UniversitySchool of Medicine approved all study procedures.

Salivary cortisol was obtained from children at T1 and T3: collection rates atboth waves were 99%. As a result of our diagnostic criteria, exclusion criteria, andoutlier assessment of children's cortisol data (discussed below), cortisol samplesincluded in our analyses were: (1) N¼195 in the analysis of T1 cortisol within T1group status, (2) N¼122 in the analysis of T3 cortisol within T3 group status,(3) N¼194 in the analysis of T1 cortisol within a history of group status, and(4) N¼127 in the analysis of T3 cortisol within a history of group status.

2.2. Diagnostic assessment

The Preschool Age Psychiatric Assessment (PAPA) was used to assess age-appropriate symptom manifestations of Axis-I psychiatric disorders at T1, T2, andT3 (Egger et al., 1999, 2003). The PAPA is a semi-structured diagnostic parentinformant interview designed for children aged 2.0–6.0 years, with establishedtest–retest reliability (Egger et al., 2006). All interviews were administered bytrained interviewers and audiotaped for later review and calibration by mastercoders. Primary caregivers were asked about their children's depressive symptomswithin the last 6 months using 31 items in the PAPA MDD module. The totalnumber of symptoms endorsed in this module was summed to provide ameasurement of children's MDD severity. All of the items in the MDD modulemapped onto the nine ‘core’ DSM-IV MDD symptom criteria. MDD status wasdetermined using a diagnostic algorithm that was consistent with DSM-IV criteria,with the exception of the 2-week duration criterion. The 2-week duration criterionwas excluded from the MDD algorithm, based on data suggesting that it may not beapplicable to preschoolers (Luby et al., 2003b; Gaffrey et al., 2011). Informationabout the durations in our MDD sample has been reported previously (Luby et al.,2009c). Diagnosis of co-morbid psychiatric disorders (i.e., mania, general anxietydisorder, posttraumatic stress disorder, separation anxiety disorder, attentiondeficit/hyperactivity disorder, oppositional defiant disorder, and/or conduct dis-order) was also assessed using the PAPA.

2.3. MDD status

Children were grouped based on T1 MDD status, T3 MDD status, and a history ofMDD status. For T1 and T3 status, children were categorized into the followinggroups: (1) healthy if they did not meet DSM-IV criteria for any psychiatric disorderand showed fewer than two DSM-IV MDD symptoms, (2) sub-syndromal MDDif they did not meet DSM-IV MDD criteria (with/without other psychiatricdisorders) but showed two or more DSM-IV-defined MDD symptoms, or (3) MDDif they meet DSM-IV MDD criteria. Seven children in the analysis of T1 group statusand nine children in the analysis of T3 group status were excluded because theymet DSM-IV criteria for any psychiatric disorders without co-morbid MDD andshowed fewer than two DSM-IV MDD symptoms.

For a history of MDD status classification, children were categorized into (1)always-healthy if they had remained healthy across the study waves (T1, T2, andT3), (2) ever sub-syndromal MDD if they had ever shown sub-syndromal MDDstatus (but not MDD) across any of the waves, or (3) ever MDD if they had everdeveloped MDD across any of the waves. Children who had not met criteria for sub-syndromal MDD or MDD but had met diagnostic criteria for other psychiatricdisorders (n¼8 in the analysis of T1 cortisol; n¼4 in the analysis of T3 cortisol)were excluded from the analyses.

2.4. Procedure

Salivary samples were collected at T1 and T3 during the course of thelaboratory assessment. Assessments started either in the morning (approximately9:00 AM) or afternoon (approximately 1:00 PM), based on family availability.To obtain saliva, children were instructed to chew on a sterile cotton roll withoutsalivary stimulant. Saliva was then frozen and stored in a vial under �20 1C.Salivary cortisol levels were later measured using the Gamma Coat CortisolRadioimmunoassay kit (DiaSovin, Stillwater, MN) at the Washington UniversityGeneral Clinical Research Center, St. Louis, MO.

The T1 and T3 assessments included three cortisol collections. The first cortisolcollection took place upon laboratory arrival, which is thought to reflect stress inanticipation of the novel laboratory environment. Then, children were separatedfrom their parent/caregiver to complete a comprehensive developmental, emo-tional, and diagnostic battery including multiple paradigms from the LaboratoryTemperament Assessment Battery (Lab-TAB), an observational measure of tem-perament (Goldsmith et al., 1995). The Lab-TAB involves several tasks designed to

H. Suzuki et al. / Psychiatry Research 210 (2013) 575–583 577

be mildly stressful for children (Gagne et al., 2011). The second collection of cortisoloccurred during the assessment, after children completed the following Lab-TABtasks: “Transparent Box” task (at T1) or the “Storytelling” and “Picture Tearing” tasks(at T3). The third cortisol collection took place at the end of the assessment, afterthe following Lab-TAB tasks: “Not Sharing,” “Box Empty,” “No Candy,” and “Impos-sible Circles” (at T1) or the “Wrong Gift” (at T3) (see Supplementary Material 1 for adescription of each Lab-TAB task). Because a similar Lab-TAB stress paradigm wasadministered across the three annual waves, by T3, subjects had become familiarwith our laboratory environment and these types of stress paradigms.

Primary caregivers' reports of the number of stressful/traumatic life eventsexperienced by their child at T1, T2, and T3 were also obtained using the PAPA(Egger et al., 1999, 2003). Information on life events was obtained using dichot-omous questions that asked whether or not the child experienced any of 15stressful events (e.g., new siblings) and 12 traumatic events (e.g., death of arelative).

2.5. Exclusion criteria

We excluded children who had lost a tooth, experienced a major acute stressor(e.g., car accident) within 24 h of the assessment, used steroids, and/or had fever(body temperature≥99.5 1F) because these experiences are known to alter cortisol(Luby et al., 2003a; Schreiber et al., 2006; Fisher et al., 2011). At T1, 82 childrenwere excluded because of tooth loss (n¼7), recent acute stress (n¼21), steroid use(n¼15), fever (n¼5), and/or missing data (n¼39). At T3, 106 children wereexcluded because of tooth loss (n¼39), recent acute stress (n¼33), steroid use(n¼20), fever (n¼5), and/or missing data (n¼21).

2.6. Statistical analyses

A common logarithmwas used to normalize skewed cortisol values. Independent-samples t-tests showed that log10-transformed cortisol levels in the morning weresignificantly higher than those in the afternoon at T1 (t(196)¼3.28 for the first,t(196)¼2.17 for the second, t(196)¼2.11 for the third, po0.05) and at T3 (t(126)¼2.80for the first, t(126)¼2.00 for the second, t(126)¼2.91 for the third, po0.05). Becauseof the differences in cortisol between morning and afternoon, we examined outliersseparately for children in the morning versus afternoon assessment, using Tukey

Table 1Characteristics of T1 baseline group status (N¼195).

Variable Healthy

Age in years (S.D.) 4.29 (0.7

Gendera Girl 35Boy 38

Ethnicitya European American 47African American 18Hispanic 0Mixed or other 8

Total household income ($)a ≤20,000 1220,001–40,000 440,001–60,000 13≥60,001 37Refused to answer 7

Caregiver's educationa High school diploma 8Some college 204-year college degree 16Graduate or higher education 29

Caregiver's marital statusa Married 50Widowed 1Separated 1Divorced 3Never married 17Refused to answer 1

Number of life event types (S.D.) Stress 3.22 (1.9Trauma 1.26 (1.0Stress and trauma 4.48 (2.5

MDD severity (S.D.) 0.81 (0.9

Note: Subjects who exhibited a psychiatric disorder and less than two MDD symptomsanalysis.

a Data are presented as frequency.n po0.05nn po0.01

boxplots. Any children whose log10-transformed cortisol values were more than threeinterquartile ranges (IQRs) above the third quartile or more than three IQRs below thefirst quartile were considered outliers (Frigge et al., 1989).

After removing outliers (n¼3 in T1 cortisol analyses; n¼8 in T3 cortisol analyses),four repeated-measures MANOVAs were performed with log10-transformed cortisol asthe dependent variable, cortisol collection sequence (first, second, and third) as thewithin-subjects variable, MDD group status (T1, T3, or history) as the between-subjectsvariable, and time of day (morning/afternoon), food intake prior to the assessment(yes/no), co-morbid psychiatric disorders (presence/absence), and the total number ofstressful and traumatic life events as the control variables. The first analysis examinedT1 cortisol reactivity in relation to T1 group status; the second analysis examined T3cortisol reactivity in relation to T3 group status; finally, the third and fourth analysesexamined T1 and T3 cortisol reactivity in relation to a history of group status. If aninteraction was significant, Bonferroni-adjusted post hoc comparisons were used toidentify specific differences.

As an additional analysis, we also run full information maximum likelihood(FIML) to impute missing cortisol data and then ran a three-way repeated-measures MANOVA where an assessment wave (T1 or T3) was additionallyincluded. The purpose of this analysis was to check our MANOVA results aftercontrolling Type II error rate, given that relatively many subjects had missingcortisol data at T1, T3, or both assessments due to our diagnostic criteria, exclusioncriteria, outlier, etc. For detailed methods and results of these imputed data, seeSupplementary Material 2.

3. Results

3.1. Background characteristics

Table 1 shows demographic and clinical characteristics forour child participants who were classified into either the healthy,sub-syndromal MDD, or MDD group at only T1 and had valid T1cortisol data. The MDD group was older and had caregiverswith lower income and education levels than the healthygroup (po0.05). However, children's age was not significantly

(n¼73) Sub-syndromal MDD (n¼76) MDD (n¼46) F or χ2

9) 4.43 (0.79) 4.69 (0.81) 3.56n

38 16 2.9238 30

42 22 5.6226 171 07 7

15 13 16.15n

11 1114 632 114 5

12 9 18.36nn

30 2519 615 6

44 18 16.420 01 26 725 190 0

9) 3.37 (1.86) 3.35 (1.85) 0.134) 1.29 (1.25) 1.61 (1.29) 1.405) 4.66 (2.45) 4.96 (2.62) 0.50

7) 3.87 (1.56) 8.41 (3.71) 180.33nn

at T1 (n¼7) were not shown in this table because they were not included in the

H. Suzuki et al. / Psychiatry Research 210 (2013) 575–583578

correlated with cortisol at T1 (p40.05), and repeated-measuresMANOVAs revealed that there were no differences in T1cortisol between household income levels or caregiver's educationlevels. Because children's age and caregivers' income and educa-tion levels did not affect cortisol at T1, these variables were notconsidered as additional covariates in the subsequent analyses.Table 2 describes background characteristics for all children whowere categorized into the healthy, sub-syndromal MDD, orMDD group at only T3 and had valid T3 cortisol data. Therewere no group differences in any demographic characteristics.Thus, no additional variables were controlled for in the analysis ofT3 cortisol.

Table 3 summarizes background characteristics for all childrenwho were always healthy or had ever shown sub-syndromal MDDor MDD at T1, T2, and/or T3 and who had valid cortisol data atleast T1 or T3. The ever MDD group had more traumatic lifeevents, more unmarried caregivers, and lower caregiver educationlevels than the ever sub-syndromal MDD and the always-healthygroups (po0.05). Nevertheless, the average number of traumaticlife events was not significantly correlated with cortisol at T1 andT3, and repeated-measures MANOVAs yielded that there were nodifferences in T1 and T3 cortisol between caregiver's marital statusor education levels. Based on this, the average traumatic life eventsand caregivers' marital status and education levels were notincluded as additional covariates.

Tables 1–3 also confirm that the MDD (or ever MDD) groupscored higher on MDD severity than the other two groups, and thesub-syndromal MDD (or ever sub-syndromal MDD) group showeda higher MDD severity score than the healthy (or always-healthy)group (po0.01). Co-morbidity rates in the MDD group were 67%at T1, 75% at T3, and 72% through all three assessments.

Table 2Characteristics of T3 follow-up group status (N¼122).

Variable Healthy

Age in years (S.D.) 6.50 (0.7

Gendera Girl 24Boy 28

Ethnicitya European American 34African American 14Hispanic 0Mixed or other 4

Total household income ($)a ≤20,000 520,001–40,000 840,001–60,000 10≥60,001 27Refused to answer 2

Caregiver's educationa High school diploma 5Some college 134-year college degree 17Graduate or higher education 17

Caregiver's marital statusa Married 34Widowed 0Separated 0Divorced 5Never married 12Refused to answer 1

Number of life event types (S.D.) Stress 1.29 (0.8Trauma 0.54 (0.6Stress and trauma 1.83 (1.1

MDD severity (S.D.) 0.87 (0.9

Note: Subjects who exhibited a psychiatric disorder and less than two MDD symptomsanalysis.

a Data are presented as frequency.nn po0.01.

Finally, Table 4 shows the attrition analysis comparing back-ground characteristics within subjects who had valid T1 cortisoldata. Overall, childrenwere more likely to drop out at T3 follow-upif they had lower family income and increased MDD severity at T1(po0.05). However, within each of the healthy, sub-syndromalMDD, and MDD groups at T1, there were no differences in anybackground characteristics between subjects included at T3 andthose excluded at T3. None of the T1 groups showed a change in, atleast, age, gender, household income, caregiver's education, care-giver's marital status, life events, and MDD severity at thefollow up.

3.2. Group status and cortisol reactivity at T1 baseline

After controlling for time of day, food intake, co-morbidities, andstressful and traumatic life events, a main effect of T1 cortisolcollection sequence was significant (Wilks' Λ¼0.91, F(2,187)¼8.77,po0.01; identical to results of adjusted univariate F-test). Never-theless, a main effect of T1 group status was not significant. Moreimportantly, the effect of T1 cortisol collection sequence significantlyinteracted with T1 group status (Wilks' Λ¼0.92, F(4,374)¼4.05,po0.01; identical to results of adjusted univariate F-test) in aquadratic (F(2,188)¼3.28, po0.05) fashion (see Fig. 1 for means andstandard errors). Post hoc tests yielded that, although there were nostatistically significant group differences in cortisol levels at anycollections, there were group differences in patterns of cortisolreactivity across the collections. Specifically, the healthy group showeda U-shaped cortisol reactivity curve; as Fig. 2 illustrates, this groupshowed a significant decrease in cortisol between the first and secondcollections and then a significant increase between the secondand third collections (po0.01). In contrast, although the MDD and

(n¼52) Sub-syndromal MDD (n¼54) MDD (n¼16) F or χ2

6) 6.31 (0.84) 6.81 (0.77) 2.51

27 4 3.1627 12

32 11 8.8415 20 17 2

9 2 8.887 18 430 70 2

5 2 1.6419 514 416 5

30 8 2.890 00 04 217 53 1

9) 1.67 (1.64) 1.69 (1.40) 1.234) 0.63 (0.85) 0.44 (0.63) 0.482) 2.30 (1.86) 2.13 (1.71) 1.21

7) 3.63 (1.52) 8.13 (2.68) 144.99nn

at T3 (n¼9) were not shown in this table because they were not included in the

Table 3Characteristics of a history of group status (N¼195).

Variable Always-healthy (n¼27) Ever sub-syndromal MDD (n¼99) Ever MDD (n¼69) F or χ2

Age in years (S.D.) 4.32 (0.85) 4.34 (0.76) 4.62 (0.81) 2.99

Gendera Girl 16 48 27 3.43Boy 11 51 42

Ethnicitya European American 19 55 34 7.41African American 6 35 22Hispanic 0 0 1Mixed or other 2 9 12

Total household income ($)a ≤20,000 4 16 20 15.0220,001–40,000 2 10 1440,001–60,000 4 20 10≥60,001 16 45 19Refused to answer 1 8 6

Caregiver's educationa High school diploma 3 15 11 16.49n

Some college 8 32 364-year college degree 3 25 12Graduate or higher education 13 27 10

Caregiver's marital statusa Married 21 60 29 24.32nn

Widowed 1 0 0Separated 0 1 3Divorced 1 5 10Never married 4 33 26Refused to answer 0 0 1

Average number of life event types (S.D.) Stress 2.19 (1.37) 2.31 (1.51) 2.52 (1.30) 0.69Trauma 0.68 (0.43) 0.82 (0.66) 1.12 (0.99) 4.40n

Stress and trauma 2.87 (1.64) 3.13 (1.81) 3.63 (1.99) 2.25

Average MDD severity (S.D.) 0.43 (0.46) 2.59 (1.28) 6.38 (3.42) 90.37nn

Note: Each group size indicates the maximum number of subjects in the group whose cortisol data were valid at least for T1 or T3. Subjects who were not categorized into thehealthy, sub-syndromal MDD, or MDD group at any assessment waves were not shown in this table because they were not included in the analysis. Values for all variables,except for life events and MDD severity, are based on T1 baseline assessment. Scores for life events and MDD severity were averaged over T1, T2, and T3.

a Data are presented as frequency.n po0.05nn po0.01.

H. Suzuki et al. / Psychiatry Research 210 (2013) 575–583 579

sub-syndromal MDD groups also showed a significant decrease incortisol between the first and second collections (po0.01), theircortisol levels did not change between the second and third collectionsand remained significantly lower than their initial levels (po0.05).

3.3. Group status and cortisol reactivity at T3 follow-up

After time of day, food intake, co-morbidities, and life eventswere controlled for, there were no main effects of T3 cortisolcollection sequence and T3 group status, and there was nosignificant interaction between them, on log10-transformed corti-sol values.

3.4. History of group status and cortisol reactivity at T1 and T3

The first analysis examined T1 cortisol reactivity in relation to ahistory of group status, with controlling for time of day, foodintake, co-morbidities, and life events. A main effect of T1 cortisolcollection sequence was significant (Wilks' Λ¼0.93, F(2,187)¼7.62,po0.01; identical to the results of the adjusted univariate F-test)in a U-shaped curve (F(1,188)¼6.47, po0.05). However, a maineffect of a history of group status and the interaction between T1cortisol collection sequence and a history of group status were notsignificant.

The next analysis examined T3 cortisol reactivity in relation toa history of group status, with controlling for time of day, foodintake, co-morbidities, and life events. Note that the sample size inthis analysis was smaller than that in the previous analysis of T1cortisol reactivity and a history of group status because thenumber of valid cases for T3 cortisol reactivity was fewer than

that for T1 cortisol reactivity. Main effects of the T3 cortisolcollection sequence and a history of group status were notsignificant, but there was a significant interaction between T3cortisol collection sequence and a history of diagnostic groupstatus (Wilks' Λ¼0.92, F(4,240)¼2.56, po0.05; identical to theresults of the adjusted univariate F-test) in a linear (F(2,121)¼4.32,po0.05) fashion (see Fig. 3 for means and standard errors). Posthoc tests showed that there were no group differences in cortisollevels at any of the collections, whereas there were groupdifferences in patterns of cortisol reactivity at T3. Fig. 4 illustratesthat the always-healthy group did not show a change in cortisollevels across the T3 collections. In contrast, the ever MDD and sub-syndromal MDD groups showed reduced cortisol levels betweenthe first and second collections (po0.01) and then did not changebetween the second and third collections; they exhibited lowercortisol levels at the third collection than their initial levels(po0.05).

4. Discussion

The present study examined cortisol reactivity across twomildly stressful laboratory assessments one year apart, amongthe three groups of children: MDD, sub-syndromal MDD, andhealthy. At T1 (baseline), all children showed decreased cortisollevels between the first and second collections, but the patterns ofT1 cortisol reactivity differed between the second and thirdcollections, depending on T1 group status. That is, healthy childrenshowed increased cortisol responses between the second andthird collections, whereas children with MDD or sub-syndromal

Table 4Characteristics of subjects included and excluded in T3 cortisol analysis (N¼202).

Variable Included at T3 (n¼89) Excluded at T3 (n¼113) F or χ2

Age in years (S.D.) 4.51 (0.84) 4.36 (0.76) 1.72

Gendera Girl 39 54 0.32Boy 50 59

Ethnicitya European American 55 58 2.83African American 25 39Hispanic 0 1Mixed or other 9 15

Total household income ($)a ≤20,000 10 31 11.47n

20,001–40,000 12 1440,001–60,000 19 16≥60,001 43 40Refused to answer 5 12

Caregiver's educationa High school diploma 10 22 5.56Some college 31 464-year college degree 19 22Graduate or higher education 29 23

Caregiver's marital statusa Married 53 61 2.88Widowed 0 1Separated 2 2Divorced 7 10Never married 26 39Refused to answer 1 0

Life events (S.D.) Stress 3.04 (1.73) 3.48 (1.99) 2.64Trauma 1.25 (1.03) 1.42 (1.29) 1.12Stress and trauma 4.29 (2.12) 4.90 (2.75) 3.00

MDD severity (S.D.) 3.12 (2.73) 4.18 (4.05) 4.44n

Note: This table includes all subjects who had valid T1 cortisol data. Values for all variables are based on T1 baseline assessment.a Data are presented as frequency.n po0.05.

Fig. 1. Patterns of baseline (T1) cortisol reactivity in children with baseline groupstatus. Each group size was: n¼73 healthy children, n¼76 sub-syndromal MDDchildren, and n¼46 MDD children. Error bars represent standard error of the mean.

Fig. 2. Baseline (T1) cortisol changes in children with baseline group status. Eachgroup size was: n¼73 healthy children, n¼76 sub-syndromal MDD children, andn¼46 MDD children. Error bars represent standard error of the mean. *Bonferroni-adjusted po0.05; **Bonferroni-adjusted po0.01.

H. Suzuki et al. / Psychiatry Research 210 (2013) 575–583580

MDD showed no change in cortisol between those collections (seeFigs. 1 and 2).

At the T3 follow-up, when children were familiar with thelaboratory environment and the stress paradigms, the patterns ofcortisol reactivity differed from those at T1 as a function of groupstatus history. Children who were always healthy did not showchanges in cortisol levels over the collections at T3 (see Figs. 3 and4), which was different from cortisol reactivity patterns shown inthe T1 healthy group. In contrast, children with ever MDD or sub-syndromal MDD status at any wave showed reduced cortisol levelsbetween the first and second collections and then showed nochange in cortisol between the second and third collections at T3

(see Figs. 3 and 4), which was the same as cortisol reactivitypatterns shown in the T1 MDD or sub-syndromal MDD groups.

4.1. Cortisol reactivity in healthy children

Healthy children initially showed a U-shaped cortisol reactivitycurve in response to mild stress, which was consistent with priorfindings in similarly aged cohorts (Luby et al., 2003a; Doughertyet al., 2010, 2011b). Therefore, for normally developing children,exposure to the novel laboratory environment was experienced asa stressor, as were exposures to the frustrating Lab-TAB tasks.Results of the follow-up analysis demonstrated that the cortisolreactivity curve became flattened (compared to baseline). Notethat this was evident only in children who were always healthy

Fig. 3. Patterns of 24-month follow-up (T3) cortisol reactivity in children withlongitudinal group status. Each group size was: n¼14 healthy children, n¼73 sub-syndromal MDD children, and n¼41 MDD children. Error bars represent standarderror of the mean.

Fig. 4. 24-month follow-up (T3) cortisol changes in children with a history ofgroup status. Each group size was: n¼14 healthy children, n¼73 sub-syndromalMDD children, and n¼41 MDD children. Error bars represent standard error of themean. *Bonferroni-adjusted po0.05; **Bonferroni-adjusted po0.01.

H. Suzuki et al. / Psychiatry Research 210 (2013) 575–583 581

across all of the study waves. It suggests that the change in cortisolreactivity between T1 and T3 within healthy children may reflectacclimation to the familiar stressful situations. That is, hypotha-lamic–pituitary–adrenal (HPA) axis functioning may respond tostress sensitively at T1 baseline, and then may adapt to the familiarstress at T3 follow-up in normally developing children. Therationale behind this idea has suggested that healthy individualsare capable of modulating their HPA axis response so that they canadapt to the demands of the environment (Juster et al., 2010;McEwen and Gianaros, 2010). In contrast, if this healthy capabilityis disturbed (e.g., due to MDD episodes), cortisol response underthe familiar stressful situations may reflect what is called ‘lack ofadaptation’, reflecting a failure to habituate to stressors (McEwen,1998; Juster et al., 2010). Nevertheless, our findings should beinterpreted with caution due to the small size of the always-healthy group (n¼14), raising the possibility of a Type II error. Inaddition, longitudinal analysis of cortisol reactivity within healthysubjects is further needed.

4.2. Cortisol reactivity in children with MDD/sub-syndromal MDD

Depressed children also experienced stress from the novelenvironment and then decreased their cortisol levels. However,unlike healthy children, their cortisol did not increase followingmild stressors, a pattern that has been referred to as bluntedcortisol reactivity (Burke et al., 2005). The pattern of these cortisolresponses again emerged at the follow-up in children with a prior

history of MDD. Notably, this is different from the patterns ofcortisol reactivity evident in healthy children, who showedchanges between T1 and T3 possibly due to their acclimation tothe stressors. Thus, childhood MDD may be characterized by(1) persistently blunted cortisol reactivity to mild stressors and(2) a failure to acclimate to familiar stressful situations. Interest-ingly, evidence for similarly altered cortisol responses was alsofound in children with sub-syndromal MDD, suggesting thatblunted cortisol reactivity and acclimation failure may manifesteven in prodromal MDD.

Blunted cortisol reactivity to stress has been consistentlyreported in MDD across the age span (Burke et al., 2005; Lopez-Duran et al., 2009), especially in severe MDD (Harkness et al.,2011). The mechanism underlying blunted cortisol reactivity mayresult from down-regulation of the HPA axis (Gunnar and Vazquez,2001). While individuals are capable of adapting to acute stress byactivating HPA axis activity, chronic stress repeatedly over-activates and eventually impairs the HPA axis (McEwen andStellar, 1993; Juster et al., 2010). As Table 3 illustrates, becausedepressed children often report more traumatic life events thanhealthy controls (Luby et al., 2009c), they may experience HPA axisdown-regulations, resulting in altered cortisol responses to stress-ful situations, such as blunted responses and a lack of acclimationto familiar stressors.

While blunted cortisol reactivity in depressed children in ourstudy was consistent with previous findings, it was unexpectedthat cortisol levels never heightened following stressors. Priorstudies employing the same stress paradigm showed thatdepressed preschoolers had persistently elevated cortisol levelsin response to stressors (Luby et al., 2003a, 2004). The discrepancyin these findings may be the result of different methodologies.Compared to the prior studies assessing cortisol at 30-min inter-vals, our study sampled cortisol at 70-min intervals, which mightbe long enough to allow depressed children to recover from eachstressor. Future research designs should assess multiple cortisollevels at shorter time intervals to clarify this discrepancy.

Moreover, children in our study did not show evidence ofhypercortisolemia, often reported in MDD (Kaufman et al., 2001;Lopez-Duran et al., 2009; Stetler and Miller, 2011). Meta-analysishas suggested that stress cortisol reactivity levels are higher inMDD than healthy controls (Lopez-Duran et al., 2009), but thismay be marked only in the afternoon (Burke et al., 2005). Thepresent study collected cortisol samples in the morning andafternoon, which might have confounded this issue. While weadjusted all analyses for assessment time, mixing cortisol samplesacross different assessment times was a limitation to our study. Or,there may be developmental differences in cortisol reactivitybecause of delayed development of neurotransmitter contentand synthetic activity (Kaufman et al., 2001). In fact, basal anddiurnal cortisol levels vary during the life span (Kaufman et al.,2001; Shirtcliff and Essex, 2008; Shirtcliff et al., 2012), which mayaffect developmental variations in cortisol levels in response tostress.

Alternatively, the extant literature suggests that hypercortiso-lemia may be specific to melancholic/anhedonic MDD in children(Luby et al., 2003a, 2004), whereas atypical MDD or MDD with co-morbid stress-related disorders may be characterized by low levelsof cortisol (hypocortisolemia) (Heim et al., 2000a; Fries et al.,2005). Furthermore, adverse experiences in childhood may mod-erate the relationship between high MDD severity and low cortisolreactivity to stress in adolescents (Harkness et al., 2011), as well asschool-aged children (Badanes et al., 2011). Future research shouldcompare cortisol reactivity between these different subtypes ofMDD accounting for key variables.

An important limitation of our study was that cortisol was notsampled at the same time of day across all subjects and all study

H. Suzuki et al. / Psychiatry Research 210 (2013) 575–583582

waves. Assessing cortisol at different times of day across the studywaves led to considerable within-subjects variability in long-itudinal cortisol levels, making it necessary to conduct cortisolanalyses at T1 and T3 separately. Future studies should bedesigned to collect saliva samples in the same manner across allsubjects, as well as across all waves of the study, wheneverfeasible. Furthermore, another limitation of the present studywas to sample cortisol only three times over 140 min, which wasless frequent than other cortisol studies (Rao et al., 2008; Kryskiet al., 2011), without a recovery period in our assessments. If, forinstance, cortisol was assessed every 10 min after each stressfulsituation and a recovery period, the patterns of cortisol responsesmay be different. Another study limitation was due to attritionbetween T1 and T3. In particular, the always-healthy group had onlyn¼14 in T3 analysis of cortisol, leading to low statistical power.While attrition in longitudinal studies is common, our results mustbe interpreted with caution due to the attrition. Finally, we did notinclude a psychiatric control group (e.g., anxiety disorders withoutMDD) or temperament control group (e.g., shyness without MDD)in order to clarify whether our findings in depressed children werespecific to MDD. We controlled for co-morbidities in all analyses,but future research should also compare cortisol reactivity betweenMDD and the additional control groups.

The present study suggests that blunted cortisol reactivity tostress might be evident in early childhood forms of MDD. More-over, persistently blunted cortisol responses to mild stressors, aswell as a failure of acclimation to familiar stressful situations, maybe early markers of childhood MDD and sub-syndromal MDDalthough further longitudinal research is needed. As such, theymay provide clues to early developmental pathophysiology pro-cesses in the disorder and may also serve as future targets for earlyintervention or prevention.

Acknowledgments

This study was supported by grant MH064769 (JLL) from theNational Institute of Mental Health. Hideo Suzuki's time wassupported by grant MH090786 (JLL) and Andy Belden's time wassupported by MH090515.

Appendix A. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.psychres.2013.06.038.

References

Ashman, S.B., Dawson, G., Panagiotides, H., Yamada, E., Wilkinson, C.W., 2002. Stresshormone levels of children of depressed mothers. Development and Psycho-pathology 14, 333–349.

Badanes, L.S., Watamura, S.E., Hankin, B.L., 2011. Hypocortisolism as a potentialmarker of allostatic load in children: associations with family risk andinternalizing disorders. Development and Psychopathology 23, 881–896.

Braddock, L., 1986. The dexamethasone suppression test. Fact and artefact. BritishJournal of Psychiatry 148, 363–374.

Brennan, P.A., Pargas, R., Walker, E.F., Green, P., Newport, D.J., Stowe, Z., 2008.Maternal depression and infant cortisol: influences of timing, comorbidity andtreatment. Journal of Child Psychology and Psychiatry 49, 1099–1107.

Bufferd, S.J., Dougherty, L.R., Carlson, G.A., Rose, S., Klein, D.N., 2012. Psychiatricdisorders in preschoolers: continuity from ages 3 to 6. American Journal ofPsychiatry 169, 1157–1164.

Burke, H.M., Davis, M.C., Otte, C., Mohr, D.C., 2005. Depression and cortisolresponses to psychological stress: a meta-analysis. Psychoneuroendocrinology30, 846–856.

Dougherty, L.R., Bufferd, S.J., Carlson, G.A., Dyson, M., Olino, T.M., Durbin, C.E., Klein,D.N., 2011a. Preschoolers' observed temperament and psychiatric disorders

assessed with a parent diagnostic interview. Journal of Clinical Child andAdolescent Psychology 40, 295–306.

Dougherty, L.R., Klein, D.N., Congdon, E., Canli, T., Hayden, E.P., 2010. Interactionbetween 5-HTTLPR and BDNF Val66Met polymorphisms on HPA axis reactivityin preschoolers. Biological Psychology 83, 93–100.

Dougherty, L.R., Klein, D.N., Rose, S., Laptook, R.S., 2011b. Hypothalamic–pituitary–adrenal axis reactivity in the preschool-age offspring of depressed parents:moderation by early parenting. Psychological Science 22, 650–658.

Egger, H., Ascher, B., Angold, A., 1999, 2003. The Preschool Age PsychiatricAssessment: Version 1.4. Center for Developmental Epidemiology, Departmentof Psychiatry and Behavioral Sciences, Duke University Medical Center,Durham, NC.

Egger, H.L., Angold, A., 2006. Common emotional and behavioral disorders inpreschool children: presentation, nosology, and epidemiology. Journal of ChildPsychology and Psychiatry 47, 313–337.

Egger, H.L., Erkanli, A., Keeler, G., Potts, E., Walter, B.K., Angold, A., 2006. Test–retestreliability of the Preschool Age Psychiatric Assessment (PAPA). Journal of theAmerican Academy of Child and Adolescent Psychiatry 45, 538–549.

Essex, M.J., Klein, M.H., Cho, E., Kalin, N.H., 2002. Maternal stress beginning ininfancy may sensitize children to later stress exposure: effects on cortisol andbehavior. Biological Psychiatry 52, 776–784.

Feldman, R., Granat, A., Pariente, C., Kanety, H., Kuint, J., Gilboa-Schechtman, E.,2009. Maternal depression and anxiety across the postpartum year and infantsocial engagement, fear regulation, and stress reactivity. Journal of the Amer-ican Academy of Child and Adolescent Psychiatry 48, 919–927.

Fisher, P.A., Van Ryzin, M.J., Gunnar, M.R., 2011. Mitigating HPA axis dysregulationassociated with placement changes in foster care. Psychoneuroendocrinology36, 531–539.

Fries, E., Hesse, J., Hellhammer, J., Hellhammer, D.H., 2005. A new view onhypocortisolism. Psychoneuroendocrinology 30, 1010–1016.

Frigge, M., Hoaglin, D.C., Iglewicz, B., 1989. Some implementations of the Boxplot.American Statistician 43, 50–54.

Gaffrey, M.S., Belden, A.C., Luby, J.L., 2011. The 2-week duration criterion andseverity and course of early childhood depression: implications for nosology.Journal of Affective Disorders 133, 537–545.

Gagne, J.R., Van Hulle, C.A., Aksan, N., Essex, M.J., Goldsmith, H.H., 2011. Derivingchildhood temperament measures from emotion-eliciting behavioral episodes:scale construction and initial validation. Psychological Assessment 23, 337–353.

Gillespie, C.F., Nemeroff, C.B., 2005. Hypercortisolemia and depression. Psychoso-matic Medicine 67 (Suppl. 1), S26–28.

Goldsmith, H.H., Reilly, J., Lemery, K.S., 1995. Laboratory Temperament AssessmentBattery: Preschool Version.

Gotthardt, U., Schweiger, U., Fahrenberg, J., Lauer, C.J., Holsboer, F., Heuser, I., 1995.Cortisol, ACTH, and cardiovascular response to a cognitive challenge paradigmin aging and depression. American Journal of Physiology 268, R865–873.

Gunnar, M.R., Vazquez, D.M., 2001. Low cortisol and a flattening of expecteddaytime rhythm: potential indices of risk in human development. Developmentand Psychopathology 13, 515–538.

Harkness, K.L., Stewart, J.G., Wynne-Edwards, K.E., 2011. Cortisol reactivity to socialstress in adolescents: role of depression severity and child maltreatment.Psychoneuroendocrinology 36, 173–181.

Heim, C., Ehlert, U., Hellhammer, D.H., 2000a. The potential role of hypocortisolismin the pathophysiology of stress-related bodily disorders. Psychoneuroendocri-nology 25, 1–35.

Heim, C., Newport, D.J., Heit, S., Graham, Y.P., Wilcox, M., Bonsall, R., Miller, A.H.,Nemeroff, C.B., 2000b. Pituitary–adrenal and autonomic responses to stress inwomen after sexual and physical abuse in childhood. Journal of the AmericanMedical Association 284, 592–597.

Juster, R.P., McEwen, B.S., Lupien, S.J., 2010. Allostatic load biomarkers of chronicstress and impact on health and cognition. Neuroscience and BiobehavioralReviews 35, 2–16.

Kaufman, J., Martin, A., King, R.A., Charney, D., 2001. Are child-, adolescent-, andadult-onset depression one and the same disorder? Biological Psychiatry 49,980–1001.

Kryski, K.R., Smith, H.J., Sheikh, H.I., Singh, S.M., Hayden, E.P., 2011. Assessing stressreactivity indexed via salivary cortisol in preschool-aged children. Psychoneur-oendocrinology 36, 1127–1136.

Lavigne, J.V., Lebailly, S.A., Hopkins, J., Gouze, K.R., Binns, H.J., 2009. The prevalenceof ADHD, ODD, depression, and anxiety in a community sample of 4-year-olds.Journal of Clinical Child and Adolescent Psychology 38, 315–328.

Lopez-Duran, N.L., Kovacs, M., George, C.J., 2009. Hypothalamic–pituitary–adrenalaxis dysregulation in depressed children and adolescents: a meta-analysis.Psychoneuroendocrinology 34, 1272–1283.

Luby, J., Belden, A., Sullivan, J., Hayen, R., McCadney, A., Spitznagel, E., 2009a. Shameand guilt in preschool depression: evidence for elevations in self-consciousemotions in depression as early as age 3. Journal of Child Psychology andPsychiatry 50, 1156–1166.

Luby, J., Heffelfinger, A., Mrakrotsky, C., Hildebrand, T., 1999. Preschool FeelingsChecklist, St. Louis, MO. Washington University.

Luby, J.L., Belden, A.C., Pautsch, J., Si, X., Spitznagel, E., 2009b. The clinicalsignificance of preschool depression: impairment in functioning and clinicalmarkers of the disorder. Journal of Affective Disorders 112, 111–119.

Luby, J.L., Heffelfinger, A., Mrakotsky, C., Brown, K., Hessler, M., Spitznagel, E.,2003a. Alterations in stress cortisol reactivity in depressed preschoolersrelative to psychiatric and no-disorder comparison groups. Archives of GeneralPsychiatry 60, 1248–1255.

H. Suzuki et al. / Psychiatry Research 210 (2013) 575–583 583

Luby, J.L., Mrakotsky, C., Heffelfinger, A., Brown, K., Hessler, M., Spitznagel, E.,2003b. Modification of DSM-IV criteria for depressed preschool children.American Journal of Psychiatry 160, 1169–1172.

Luby, J.L., Mrakotsky, C., Heffelfinger, A., Brown, K., Spitznagel, E., 2004. Character-istics of depressed preschoolers with and without anhedonia: evidence for amelancholic depressive subtype in young children. American Journal ofPsychiatry 161, 1998–2004.

Luby, J.L., Si, X., Belden, A.C., Tandon, M., Spitznagel, E., 2009c. Preschool depres-sion: homotypic continuity and course over 24 months. Archives of GeneralPsychiatry 66, 897–905.

Luby, J.L., Sullivan, J., Belden, A., Stalets, M., Blankenship, S., Spitznagel, E., 2006. Anobservational analysis of behavior in depressed preschoolers: further validationof early-onset depression. Journal of the American Academy of Child andAdolescent Psychiatry 45, 203–212.

McEwen, B.S., 1998. Protective and damaging effects of stress mediators. NewEngland Journal of Medicine 338, 171–179.

McEwen, B.S., Gianaros, P.J., 2010. Central role of the brain in stress and adaptation:links to socioeconomic status, health, and disease. Annals of the New YorkAcademy of Sciences 1186, 190–222.

McEwen, B.S., Stellar, E., 1993. Stress and the individual. Mechanisms leading todisease. Archives of Internal Medicine 153, 2093–2101.

Nestler, E.J., Barrot, M., DiLeone, R.J., Eisch, A.J., Gold, S.J., Monteggia, L.M., 2002.Neurobiology of depression. Neuron 34, 13–25.

Pariante, C.M., Lightman, S.L., 2008. The HPA axis in major depression: classicaltheories and new developments. Trends in Neurosciences 31, 464–468.

Rao, U., Hammen, C., Ortiz, L.R., Chen, L.A., Poland, R.E., 2008. Effects of early andrecent adverse experiences on adrenal response to psychosocial stress indepressed adolescents. Biological Psychiatry 64, 521–526.

Schreiber, J.E., Shirtcliff, E., Van Hulle, C., Lemery-Chalfant, K., Klein, M.H., Kalin, N.H., Essex, M.J., Goldsmith, H.H., 2006. Environmental influences on familysimilarity in afternoon cortisol levels: twin and parent–offspring designs.Psychoneuroendocrinology 31, 1131–1137.

Shirtcliff, E.A., Allison, A.L., Armstrong, J.M., Slattery, M.J., Kalin, N.H., Essex, M.J.,2012. Longitudinal stability and developmental properties of salivary cortisollevels and circadian rhythms from childhood to adolescence. DevelopmentalPsychobiology 54, 493–502.

Shirtcliff, E.A., Essex, M.J., 2008. Concurrent and longitudinal associations of basaland diurnal cortisol with mental health symptoms in early adolescence.Developmental Psychobiology 50, 690–703.

Stetler, C., Miller, G.E., 2011. Depression and hypothalamic–pituitary–adrenalactivation: a quantitative summary of four decades of research. PsychosomaticMedicine 73, 114–126.

Wichstrom, L., Berg-Nielsen, T.S., Angold, A., Egger, H.L., Solheim, E., Sveen, T.H.,2012. Prevalence of psychiatric disorders in preschoolers. Journal of ChildPsychology and Psychiatry 53, 695–705.

Young, E.A., Lopez, J.F., Murphy-Weinberg, V., Watson, S.J., Akil, H., 2000. Hormonalevidence for altered responsiveness to social stress in major depression.Neuropsychopharmacology 23, 411–418.