The current study investigated 1) the associations between ELS, early neuroendocrine function, and adolescent resting-state fcMRI; and 2) the differential association of adolescent resting-state fcMRI, as well as ELS and early neuroendocrine function, with concurrent symptoms of anxiety and depression. To affirm that any associations observed were due to early experience, adolescent life stress and afternoon basal cortisol from this period were also examined. Our central prediction was that ELS would be associated with the development of heightened late afternoon childhood basal cortisol levels and that both ELS and childhood cortisol would predict variations in adolescent fcMRI. Specifically, we predicted that ELS and cortisol would be positively correlated, and that both would be inversely related to connectivity estimates between the amygdala and vmPFC, which would reflect basal abnormalities in this key regulatory circuit. Following from this, we also predicted that resting-state amygdala-vmPFC fcMRI would be associated with concurrent adolescent internalizing symptoms, especially anxiety. Additional analyses explored whether resting-state amygdala-vmPFC fcMRI mediated the association of childhood cortisol and internalizing symptoms. Finally, based on gender differences found in numerous studies summarized above, gender differences were investigated in all analyses.
In the present study, we acquired structural MRI and resting-state fMRI data, along with self-reported psychiatric symptoms, from 57 (28 female, Mage
: 18.44, SD
= 0.19 years) participants selected from the Wisconsin Study of Families and Work (WSFW)4
, a prospective longitudinal study of a community sample beginning during the prenatal period. FcMRI of the amygdalae at age 18 years was computed by first defining masks of the left and right amygdala using previously-developed30
probabilistic maps in Talairach space31
. The averaged preprocessed signal time-courses from each amygdala were extracted and regressed with resting-state time-courses from each voxel in the rest of the brain. Connectivity values were then regressed across participants against measures of mean salivary basal cortisol obtained in childhood at age 4.5 years, which had been collected on 3 consecutive days at a pre-selected target time between 3 and 7pm (actual collection time M
= 5:56pm, SD
= 88 min). We chose this time as most likely to reflect children’s experiences of stress during the day32
. Cortisol was then assayed using the Pantex RIA (Santa Monica, CA), modified for salivary data. Mean late afternoon basal cortisol values were log10
transformed and residualized for time of collection and concomitant over-the-counter medication usage4
. ELS exposure was measured using a composite measure of early maternal stress that was computed using maternal reports of depressive symptoms, parenting stress, marital conflict, role overload, and financial stress, averaged over assessments conducted when children were 1, 4, and 12 months old4
. Current adolescent psychiatric symptoms were indexed via self-report on the MacArthur Health and Behavior Questionnaire33
(HBQ). The general anxiety and depressive subscales were computed; the externalizing subscale was also used as a control variable in all analyses of psychiatric symptoms. Adolescent recent life stress was measured with a self-report measure derived from the Adolescent Perceived Events Scale34
and the Life Events Survey35
. Finally, all data were winsorized to within three standard deviations of the mean to normalize the distribution in the subsample.
Initial analyses examined voxel-wise functional connectivity estimates using the left amygdala seed (associations using the right amygdala were similar). Results indicated significant positive fcMRI with the contralateral amygdala, dorsomedial and ventrolateral PFC, ventral striatum, superior temporal gyrus, and vmPFC at rest, consistent with earlier studies10
ELS, Childhood Cortisol, and Adolescent fcMRI
Voxel-wise analyses with the left amygdala seed revealed no statistically significant associations between ELS and resting-state fcMRI between the amygdala and any other brain regions. However, as predicted, childhood cortisol was negatively correlated with functional connectivity estimates between the left amygdala and a voxel cluster in the vmPFC (peak t-statistic: t(56) = −5.97, R2 = 0.36, FDR-corrected p = 0.01). Higher late afternoon cortisol levels during childhood were associated with decreased amygdala-vmPFC fcMRI fourteen years later (see and ). No voxel-wise relations were detected between amygdala-vmPFC fcMRI estimates and either adolescent basal cortisol or current life stress, suggesting that the observed effects were specific to childhood late afternoon basal cortisol function, not current stress or cortisol levels.
Figure 1 Correlation between late afternoon cortisol at child age 4.5 yrs and resting-state fcMRI to the left amygdala at 18 yrs. Connectivity between the left amygdala and vmPFC is significantly negatively associated with childhood cortisol (R2 = 0.36, FDR-corrected (more ...)
Summary of Resting State fcMRI Estimates with Left Amygdala ROI as Predicted by Childhood Late Afternoon Basal Cortisol Levels.
Bivariate Pearson-r (two-tailed) correlations for the total sample revealed a significant positive association between ELS and childhood late afternoon cortisol, and a significant inverse association between childhood cortisol and adolescent amygdala-vmPFC fcMRI (). When considered separately for males and females, results suggested that these effects were largely specific to females. Moreover, only females demonstrated a detectable, but marginally-significant (p = 0.07), inverse relationship between ELS and amygdala-vmPFC fcMRI, with higher ELS exposure linked to lower functional connectivity.
Summary of Pearson-r Bivariate Correlation Statistics for ELS, Childhood Cortisol, and Adolescent Amygdala-vmPFC Functional Connectivity, Life Stress and Cortisol
These results, and the temporal ordering of the variables, suggested that the associations of ELS and childhood cortisol with adolescent amygdala-vmPFC fcMRI might reflect a moderated mediational process. Thus, we constructed a structural equation model using Mplus36
(version 5.2) to determine if childhood cortisol mediated the association between ELS and amygdala-vmPFC fcMRI, and whether gender moderated this association. In addition, adolescent recent life stress and current afternoon basal cortisol were included as controls to confirm that the observed effects were indeed specific to early stress and childhood basal cortisol function. This model () showed excellent fit and accounted for 39.1% of the variance in fcMRI. There was no direct effect of ELS on adolescent amygdala-vmPFC fcMRI, and the mediating effect of childhood cortisol was marginally significant (Indirect Est = −0.06, p
= 0.09). Further, gender significantly moderated both pathways. For females only, higher levels of ELS predicted increased levels of childhood afternoon basal cortisol, which, in turn, predicted decreased amygdala-vmPFC functional connectivity.
Figure 2 Structural equation model examining the moderating effect of gender on the mediation through childhood late afternoon basal cortisol of the association between early life stress and amygdala-vmPFC fcMRI. The SEM model demonstrated good fit: Chi2 = 1.89, (more ...)
Adolescent Resting-State fcMRI and Concurrent Internalizing Symptoms
Bivariate Pearson-r (two-tailed) correlations revealed no significant associations between adolescent amygdala-vmPFC fcMRI and concurrent anxiety or depressive symptoms prior to controlling for each other and for externalizing symptoms (). However, when their covariation was considered, Pearson-r partial correlations revealed that, for the total sample, adolescent amygdala-vmPFC fcMRI was significantly inversely correlated with adolescent anxiety and significantly positively correlated with adolescent depression. When considered separately for males and females, results suggested, again, that these effects were largely specific to females. Further, significant associations between childhood cortisol and adolescent internalizing symptoms suggested that amygdala-vmPFC fcMRI might play a mediating role, especially for females.
Summary of Pearson-r Bivariate and Partial Correlation Statistics for Adolescent Anxiety and Depression Symptoms with ELS, Childhood Cortisol, and Adolescent Amygdala-vmPFC Resting-state Functional Connectivity
We constructed a second moderated mediation SEM to examine whether adolescent amygdala-vmPFC fcMRI was a mediator in the association of childhood cortisol and adolescent anxiety symptoms, and whether this process was moderated by gender. This model () demonstrated good fit and accounted for 64.6% of the variance in adolescent anxiety symptoms. There was no direct effect of childhood cortisol on adolescent anxiety symptoms, and the mediating effect of adolescent amygdala-vmPFC fcMRI was marginally significant (Indirect Est = 0.13, p = 0.08). Further, gender significantly moderated the association of childhood cortisol with fcMRI. However, because this model revealed a non-significant moderating effect of gender on the association of fcMRI with anxiety (p = 0.23), an alternative model was constructed without this pathway. In addition, because fcMRI estimates were obtained concurrently with anxiety symptoms, and there was no temporal precedence by which to define mediation, we built a second alternative model with the positions of anxiety symptoms and fcMRI reversed. For both of these alternative models, the fit statistics were decreased and the model fit was poor (see Online Methods). Together, these results show that, especially for females, amygdala-vmPFC fcMRI is negatively correlated with concurrent symptoms of anxiety (see ). Further, for females only, the developmental pathway between childhood cortisol and adolescent anxiety symptoms operates through adolescent amygdala-vmPFC fcMRI.
Figure 3 Structural equation model examining the moderating effect of gender on the mediation through amygdala-vmPFC fcMRI of the relation between childhood late afternoon basal cortisol and adolescent anxiety. The SEM model demonstrated good fit: Chi2 = 9.95, (more ...)
Partial correlation between resting-state left amygdala-vmPFC fcMRI and concurrent self-reported anxious symptoms in adolescent females, controlling for concurrent symptoms of depression and externalizing behaviors (R2 = 0.31, p = 0.004).
To examine depression as an outcome, we built a parallel moderated mediational model. Although this model demonstrated good fit, gender did not moderate the path from fcMRI to depression and thus, that pathway was omitted (see Online Methods). This modified model demonstrated improved fit and accounted for 56.2% of the variance in depression (). There was a marginally-significant direct effect of childhood cortisol on adolescent depressive symptoms, and the mediating effect of adolescent amygdala-vmPFC fcMRI was significant (Indirect Est = −0.18, p = 0.02). Further, as with anxiety, when depressive symptoms and amygdala-vmPFC were transposed in an alternative model, the fit statistics were decreased and the model fit was poor (see Online Methods). Together, these results show that, for all adolescents, increased amygdala-vmPFC functional connectivity is associated with the development of depressive symptoms. In addition, for females only, the developmental pathway between childhood cortisol and adolescent depressive symptoms operates, in part, through adolescent amygdala-vmPFC fcMRI.
Figure 5 Structural equation model examining the moderating effect of gender on the mediation through amygdala-vmPFC fcMRI of the relation between childhood afternoon cortisol and adolescent depression. The SEM model demonstrated good fit: Chi2 = 7.56, p > (more ...)