This report includes work arising from the following clinical protocol: NCT01434368. Recruitment efforts for this longitudinal study continue and we have completed approximately 70% of our planned enrollment of 12-13 year old children and approximately 40% of our planned enrollment of prepubertal children (8 year olds). We have observed several important methodologic variables that potentially will inform future studies: First, age alone is not sufficient to determine prepubertal status since we have identified approximately 10 - 20% of girls who show advanced Tanner breast stage at age 8. Breast development was unilateral and reflected early estradiol secretion in these girls. Second, neither chronologic age nor Tanner stage are sufficient markers of sex steroid (i.e., estradiol) exposure in boys and girls, since we have identified abnormally advanced bone age relative to their chronologic age and controlled for sex (as measured by left wrist x-rays) in approximately 10% of children screened for enrollment in this study (who otherwise are within normal range for body mass index BMI). We also have documented the reversal of pubertal stage in both boys and girls during early and late puberty consistent with the original reports by Tanner (but rarely reported in the subsequent clinical literature). We are currently investigating the potential endocrine accompaniments of this observed reversal of pubertal stage. Finally, we have identified abnormally increased or decreased BMI (defined by normal BMI percentiles between 15th and 85th percentiles) which is accompanied by alterations in sex steroid secretion in approximately 17% of otherwise normal healthy children screened for this study thus emphasizing the importance of controlling for BMI in studies of the relationship between sex steroid secretion and brain development. We are currently investigating the effects of these early changes in gonadal activity as reflected by advanced bone age on brain structure and function. Preliminary results of HPA axis measures in these children in which we employ the cortisol awakening response (CAR), suggest that sex differences in CAR exist, with girls having greater HPA axis activity (i.e., higher peak cortisol and more prolonged secretion) compared with boys. However, we observed no significant main or interactive effects of pubertal stage on salivary cortisol (CAR) measures. These data are consistent with studies in animals that demonstrate increased HPA axis responsivity to a variety of stressors in females compared with males; however, our preliminary findings document that the sex-differences in humans emerge prior to puberty and therefore do not reflect the onset of gonadarche for their expression. We also have observed the emergence of abnormalities in sleep in late pubertal children compared with prepubertal children. Specifically, complaints by parents and the childs own self reports describe patterns of increased sleep latency (i.e., time to get to sleep), sleep disruption, and duration of sleep in late-puberty boys and girls. Although preliminary, we observed that these changes across puberty correlate positively with estradiol levels in both sexes albeit a small sample. Nonetheless, as we continue to follow individual children through the pubertal transition the appearance of sleep difficulties and the possible substrates of this change will be more comprehensively examined. Preliminary results of multi-modal neuroimaging studies in this project suggest that pubertal stage contributes to several measures of adolescent brain development and accounts for differences between adolescent and adult brain development that are both brain region- and sex-specific. First, we employed diffusion tensor imaging (DTI), and myelin water fraction (MWF) imaging (mcDESPOT method) to examine brain development in this sample of children. Bone age (a measure of tissue exposure to estradiol in both boys and girls) and Tanner stage were highly correlated with maturation of the corticospinal tracts as measured by both DTI and MWF. In contrast, gonadal volume and plasma steroid hormone levels were associated with minimal changes in MWF medial to the thalamus. These findings suggest that bone age and Tanner stage may co-vary with white matter maturation during the early pubertal phase, while gonadal volume and steroid hormones may be associated with more restricted changes in this early phase of development. Second, resting state functional connectivity studies have identified sex-differences in pubertal development of amygdalar resting functional connectivity. Our data suggest that biological sex influences amygdalar functional connectivity across puberty, with boys showing higher coupling between the amygdala and ventral striatum, temporal gyrus, and lateral prefrontal cortex compared with girls. However, our data also show similar amygdala medial prefrontal functional connectivity in boys and girls, suggesting that developmental alterations in amygdala connectivity are region specific and influenced by age and sex. Third, cognition-activated fMRI studies document differential working memory-related prefrontal-hippocampal functional connectivity in children (early puberty) and adolescents (late puberty). We found differential negative prefrontal-hippocampal functional coupling during working memory in children but not adolescents, which could indicate altered reciprocal coupling between an immature prefrontal cortex and the hippocampus, along with changes in the level of cooperativity between these brain regions accompanying prefrontal cortical development during adolescence. Fourth, our studies of reward-related neurocircuitry in these children show a differential recruitment of the ventral striatum and prefrontal cortex during reward processing in early and late-pubertal children. These data provide evidence for an age-dependent differential neural recruitment in the ventral striatum and prefrontal cortex in children and adolescents, in line with past studies showing differential rates of development of subcortical regions and prefrontal cortex. Efforts to distinguish between age- and hormone-related actions to mediate these findings remains unclear and awaits completion of the longitudinal (repeated measures within children) components of this study. Finally, preliminary results from this study could provide insight into developmental processes relevant for vulnerability to early life trauma and future psychiatric morbidity. Evidence suggests that the structural integrity of the uncinate fasciculus, one of the major white matter tracts connecting limbic and prefrontal regions, predicts amygdala activation to fearful faces and is abnormal in some anxiety conditions including PTSD. However, few studies have investigated the developmental trajectory of the amygdalas structural and functional connectivity. In both children and adults, we examined the relationship between structural connectivity of the uncinate fasciculus and functional activation of the amygdala during viewing of aversive emotional faces. During the viewing of aversive emotional faces, in children an increased integrity of the uncinate fasciculus predicted increased amygdala activity and functional connectivity with the anterior cingulate cortex, while this relationship was not present in adults. These results suggest that white matter integrity may impact the functioning of the fronto-amygdalar circuitry differently in children than in the fully developed adult brain.
