White matter microstructure in youth with and at risk for bipolar disorder

Abstract Objectives Bipolar disorder (BD) and familial risk for BD have been associated with aberrant white matter (WM) microstructure in the corpus callosum and fronto‐limbic pathways. These abnormalities might constitute trait or state marker and have been suggested to result from aberrant maturation and to relate to difficulties in emotion regulation. Methods To determine whether WM alterations represent a trait, disease or resilience marker, we compared youth at risk for BD (n = 36 first‐degree relatives, REL) to youth with BD (n = 36) and healthy volunteers (n = 36, HV) using diffusion tensor imaging. Results Individuals with BD and REL did not differ from each other in WM microstructure and, compared to HV, showed similar aberrations in the superior corona radiata (SCR)/corticospinal tract (CST) and the body of the corpus callosum. WM microstructure of the anterior CC showed reduced age‐related in‐creases in BD compared to REL and HV. Further, individuals with BD and REL showed in‐creased difficulties in emotion regulation, which were associated with the microstructure of the anterior thalamic radiation. Discussion Alterations in the SCR/CST and the body of the corpus callosum appear to represent a trait marker of BD, whereas changes in other WM tracts seem to be a disease state marker. Our findings also support the role of aberrant developmental trajectories of WM microstructure in the risk architecture of BD, although longitudinal studies are needed to confirm this association. Finally, our findings show the relevance of WM microstructure for difficulties in emotion regulation—a core characteristic of BD.

and regulation. 6 However, to differentiate quantitative risk factors from markers of disease progression, it is necessary to also study healthy individuals at high risk to develop bipolar disorder (ie, unaffected first-degree relatives of individuals with BD; REL). In addition, studies in individuals at risk for BD before the first peak of onset that occurs between 21-25 years 7 ) offer the possibility to identify adaptive functional changes associated with resilience and will thus further advance our etiological understanding of BD.
The aberrant development of white matter (WM) pathways interconnecting the prefrontal cortex with limbic brain regions, which previously showed aberrant activity in individuals with and at risk for BD, 8 has been argued to be central to the emergence of BD. 9 The microstructure of WM pathways can be quantified using diffusion tensor imaging (DTI) and is often described in terms of fractional anisotropy (FA), which is positively correlated with increased directional coherence, increased axon packing density, and smaller axon diameter in WM. 10 In adult BD, reduced FA in the anterior corpus callosum (CC) and the cingulum bundle has emerged as the most robust finding in several meta-analyses. 11,12 In addition, reduced FA in association fibers (eg, superior longitudinal fasciculus [SLF], anterior thalamic radiation [ATR]) and projection fibers (eg, uncinate fasciculus [UNC], corticospinal tract [CST]) has been frequently reported in adult BD (for review see Ref. 13 ). Of note, 12 studies were conducted in pediatric BD and, as in adult BD, reduced FA in the anterior CC, cingulum bundle, UNC, SLF, CST and ATR, 14 emerged as the most robust findings. Moreover, seven studies in unaffected adult REL reported reduced FA in the CC, 15 cingulum bundle, 16 SLF, 16,17 ATR, 15,17 and UNC. [15][16][17] Based on these findings, it has been argued that altered WM microstructure might represent a quantitative risk marker for BD, meaning it plays an antecedent, possibly causal, role in the pathophysiology of BD. 13 However, the two studies that compared adolescent REL (n = 25-79, age: [15][16][17][18][19][20][21] to HV's yielded conflicting results. While one study reported reduced FA in the CC, CST, UNC, IFOF, ILF, SLF, and ATR, 15 the other found FA increases in the CC, UNC, ILF, SLF, and cingulum bundle. 18 Thus, more DTI studies in youth at risk are needed to discern whether these abnormalities truly reflect the risk for BD. In this context it is also of interest that little is known as to how and when WM abnormalities in BD develop. It has been proposed that failed pruning of axons and dendrites between early childhood and puberty, particularly in prefrontal-limbic tracts such as UNC, impair top-down emotion regulation in BD. 9 Others have proposed that alterations in the normal developmental myelination of fiber tracts, which continues until the third decade of life 19 or altered myelin plasticity in response to experiences 20 contribute to the risk for BD. 13 However, studies addressing these developmental hypotheses are scarce. A cross-sectional study implied age-related FA increases in the CC, cingulum bundle and UNC in HV, but FA decreases in BD. 21 Consistent with this observation, the absence of normative FA increases in the UNC was observed in a longitudinal study using a region of interest (ROI) approach that followed individuals with BD (n = 27) for 2.5 years during adolescence/young adulthood. 22 However, a 2-year longitudinal study could not identify differential developmental trajectories of WM in REL (n = 69) compared to HV during adolescence/young adulthood. 23 Despite these interesting findings, more studies are needed to advance our understanding how WM alterations in BD develop.
Finally, it has been hypothesized that abnormal WM microstructure contribute to emotion regulation difficulties in BD. 9 However, no studies have directly tested the link between WM microstructure and difficulties in emotion regulation, although there is some evidence that WM microstructure particularly in the anterior CC relates to symptoms of negative affect indicative of disturbed emotion regulation. 24 The present study is the first to directly compare WM microstructure in an adolescent sample of 36 individuals diagnosed with BD, 36 HV, and 36 REL to discern risk from disease and resilience markers. Based on the literature, we expected widespread FA reductions in BD and possibly less pronounced FA alterations in overlapping areas in REL. Second, we explored group*age interactions in significant tracts and the CC and UNC that were previously associated with differential developmental trajectories in BD. 21,22 Compared to HV, we expected reduced age-related FA increases in BD but not in REL. Third, we sought to determine the contribution of WM microstructure to the difficulties in different dimensions of emotion regulation, which we assessed with the Difficulties in Emotion Regulation Scale 25 (DERS).
Most of the BD subsample was euthymic; four participants met criteria for a hypomanic episode, and one met criteria for a mixed episode. BD and REL were comparable regarding depressive symptoms (P = .13), levels of irritability (P = .08) and anxiety (P = .10). BD youth showed more manic symptoms (P < .001) and lower psychological and social functioning (P < .001) than REL. The number of BD youth and REL with an anxiety disorder was comparable. ADHD was more frequently diagnosed in BD youth (P = .01). Five individuals with BD and 31 REL were unmedicated (see Table 1).
Participants over age 18 and parents of minor participants gave written informed consent after receiving a complete description of the study; minors gave written assent. Procedures were approved by the Institutional Review Board of the National Institute of Mental Health.

| Clinical assessment
All participants were assessed with the Kiddie Schedule for Affective Disorders and Schizophrenia (K-SADS) with the DMDD supplement 27 by master's and doctoral-level clinicians. A senior clinical psychologist (MAB) or psychiatrist (EL, KT) confirmed the primary diagnosis. Exclusion criteria comprised neurological disorders, autism spectrum disorders, substance use within the last two months, conditions for which MRI is contraindicated, and full-scale IQ <70, measured using the Wechsler Abbreviated Scale of Intelligence (WASI). 28 Manic and depressive symptoms were assessed by master's and doctoral-level clinicians, with excellent inter-rater reliability (κ >0.9 for all ratings) within a week of scanning in BD youth and REL using the Young Mania Rating Scale (YMRS) 29 and the Children's Depression Rating Scale (CDRS) 30 ; respectively. Irritability and anxiety symptoms were rated by both children and parents with the Affective Reactivity Index (ARI) 31

and the Screen for Child Anxiety
Related Disorders (SCARED). 32 Global functioning was assessed in BD youth and REL by master's and doctoral-level clinicians using the Children's Global Assessment Scale (CGAS). 33 Child and parent completed the DERS, 34 which has shown good internal reliability and convergent validity in adolescents. 35 The DERS  goal-directed behavior while experiencing negative emotions, and (6) the extent to which one is clear about the emotions one is experiencing. The total scale and subscales were scored to indicate the frequency with which difficulties in emotion regulation are experienced, with scores ranging from 1 ("Almost never") to 5 ("Almost always"). The mean scores from parent and child ratings are presented in Table 2.

| Processing of DTI data
Before processing, all diffusion data were visually inspected by  FA is the most widely used measure of WM microstructure.
However, it is impacted by a range of biological factors such as myelination, fiber density, axonal size, and fiber coherence. Therefore, we also projected maps of axial diffusivity (AD) and radial diffusivity (RD) onto the skeleton. Radial diffusivity is a measure of restricted diffusivity across the axonal walls describing the permeability of axonal membranes and therefore serves as an indirect estimate of the axonal myelination level. 40 Axial diffusivity, which reflects unobstructed diffusivity along the axonal axis, is more indicative of axonal organization. 41

Hypothesis 1 Alterations in WM microstructure a trait marker of BD.
To test our primary hypothesis of abnormal white matter microstructure in both BD and REL, we used the Permutation Analysis of Linear Models (PALM) 42 testing for group differences in FA, RD, and AD with age and medication load as nuisance variables. Since the location of the aberrant WM microstructure in BD and REL is a matter of debate, we chose a whole brain approach. For voxel-wise analyses, 5000 permutations were used. Results were considered significant if they passed a threshold of P < .05 using threshold-free cluster enhancement, a family-wise error rate correction for multiple comparisons across voxels, and correction over contrasts. Of note, this approach renders an omnibus-test obsolete. Further, we accounted for the fact that 10 participants were related (2 BD-REL, 3 REL-REL) using multi-level exchangeability blocks. We also used permutation-based nonparametric combination (NPC) with Fisher's combination of P-values for the joint analysis of the three diffusion metrics in an explorative analysis. 43 Results of this exploratory analysis are presented in the Supporting Information, S2. For each cluster that passed the significance threshold, the coordinates and P-values of the peak-voxel (P min ) and the cluster sizes are reported. Results were back-projected into native FA spaces ensuring that voxels fell within WM pathways for each person.

Hypothesis 2 Differential age effects on WM microstructure.
To determine whether age regression slopes differ between groups, we extracted FA values from three ROIs implicated by previous studies (ie, the genu of the CC, the left and right UNC 21,22

Hypothesis 3 WM microstructure predicts difficulties in emotion
regulation.
To address the hypothesis that WM microstructure is associ- to test for collinearity, normality, outliers, and leverage. All results were considered significant with P < .0125 thereby applying Bonferroni correction for four regression models (DERS, ARI, SCARED, CGAS).

| Statistical analysis of demographic and questionnaire data
All statistical analyses of clinical data were performed using SPSS.
For demographic data conforming to the assumptions of parametric analysis, we used either analysis of variance or Student's t test, for nominal demographic data Chi-square tests were computed. Results were considered significant with P < .05. There were no significant group differences with regard to AD and RD. However, we observed marginally higher RD in BD youth compared to HV for the cluster in the CST (x = −24, y = −20, z = 35, 2256 voxel, P min = .058).

Hypothesis 2 Differential age effects on WM microstructure.
A significant age*group interaction effect was observed in the genu and the body of the CC, indicating steeper age-related FA increases in HV and REL compared to BD (see Figure 2). There was no significant age*group interaction in the left and right uncinate fasciculus or left and right corticospinal tract.
In more detail, FA in the genu of the corpus callosum was predicted by the dummy variable coding a differential age effect in BD youth (β stand = −0.33, t = −3.54, P = .001, adjusted R 2 = .106,

Hypothesis 3 WM microstructure as predictor of emotional difficulties.
In a first step we analyzed the associations between the four criteria of the regression models (DERS, ARI, SCARED, and CGAS). We observed a positive correlation between the DERS and ARI score (r = .62, P < .001) as well as the SCARED score (r = .44, P < .001).

| D ISCUSS I ON
Compared to HV, BD youth and REL displayed reduced FA in the bilateral CST and the body of the CC and were overall comparable regarding their WM microstructure. Further, BD youth and REL showed more difficulties in emotion regulation and were more irritable than F I G U R E 2 Differential age effect in bipolar youth compared to relatives and healthy volunteers (HVs). A, Shows the association between fractional anisotropy in the genu of the corpus callosum and age for the three study groups separately. B, Illustrates the relationship between fractional anisotropy in the body of the corpus callosum and age for the cluster in the body of the corpus callosum where significant group differences between relatives and HVs were observed HVs. While the microstructure of the ATR was associated with difficulties in emotion regulation, irritability was related to FA in the anterior CC. As expected, BD youth showed more widespread reductions in FA compared to HV. Further, BD youth displayed less age-related increase in the FA of the genu and the body of the CC compared to HV and REL.

Hypothesis 1 Alterations in WM microstructure a trait marker of BD.
There were no differences in the WM microstructure between individuals with and at risk for BD. However, compared to HV, both individuals with and at risk for BD showed reduced FA in the CST and CC. In REL, but not BD youth, the findings in the CST were more pronounced in the subgroup with a diagnosis of ADHD, which suggests that abnormalities in this tract might not be specific to BD. This interpretation is consistent with the literature that comprises reports of reduced FA in the CST in individuals with 14 or at risk 15 for BD and in individuals with ADHD. 44 The CST is a major motor pathway, and its microstructure has been positively associated with processing speed. 45 Thus, altered WM microstructure in the CST might contribute to both reduced processing speed 46 in BD.
Reduced FA in the body of the CC could not be attributed to other clinical variables, which supports the idea that abnormal WM microstructure in the CC might be a trait marker of BD. Indeed, reduced FA in the CC has emerged as the most robust finding in several meta-analyses in individuals with BD 11,12 and has also been reported repeatedly in unaffected REL during late adolescence 15 and adulthood. 16 The cluster we identified in REL was located in the body of the CC known to interconnect parietal and temporal cortices. The body of the CC develops into the corona radiata, which has been associated with processing speed and working memory performance. 47 In individuals with BD, reduced FA was observed in a large cluster spanning the entire CC. Future studies should test whether aberrant microstructure in the CC relates to the reduced processing speed and impaired working memory performance previously reported in REL. 48 Unlike many studies in adult individuals with BD, 13 we were unable to attribute reductions in FA to increases in RD, which serves as an indirect estimate of axonal myelination. Thus, abnormalities in RD that are often seen in adult BD might reflect altered myelin plasticity, referring to changes in myelination in response to experiences, 20 rather than altered developmental myelination. However, more studies are needed to prove this hypothesis.

Hypothesis 2 Differential age effects on WM microstructure.
Consistent with our hypothesis, we observed the age regression slope was reduced for FA in anterior CC in BD only. 21 Further, comparable age regression slopes between REL and HV is consistent with the literature. 23 Although longitudinal studies are needed to determine the precise time course of CC maturation in BD, numerous reports of reduced FA in the anterior CC in adults with BD suggest that this is not just a developmental delay in CC maturation, but that reduced FA in the anterior CC continues to represent a disease marker in later life.
Of note, we were unable to replicate findings of differential age effects in the CST 21 and UNC 21,22 previously reported in BD youth.
It might also be that such effects are only detectable in late adolescence/young adulthood, 22 particularly in tracts such as the UNC, where age-related FA increases do not plateau until the 3rd decade of life. 19 Further, we might have been unable to detect these effects using a cross-sectional as opposed to a longitudinal design and a TBSS-as opposed to a tractographic approach, which might be more sensitive to developmental effects. 21,22 Hypothesis 3 WM microstructure predicts emotional difficulties.
In contrast to our hypothesis, FA in the ATR, but not in the UNC, was associated with difficulties in emotion regulation, which appear to not be specific to BD or the risk for BD as they were also associated with the diagnosis of anxiety disorders. The ATR is a major projection from the thalamus that carries reciprocal connections from the striatum to the frontal cortex. 49 The FA in the right ATR has been previously associated with reduced learning from unexpected negative feedback (negative prediction error) and increased risk-taking. 17 With regard to emotion regulation, data suggest that during F I G U R E 3 Partial regression plots of fractional anisotropy in (A) the right and (B) the left anterior thalamic radiation and difficulties in emotion regulation as dependent variable and (C) fractional anisotropy in the genu of the corpus callosum and irritability across the entire sample cognitive emotion regulation (ie, distraction, reappraisal) the PFC is able to reduce the impact of the hippocampus, amygdala, and ventral tegmental area on ventral striatal negative prediction error signals, allowing for flexible adaptation of behavior. 50 Changes in the microstructure of the ATR connecting the PFC with the striatum might impair this flexibility.
Surprisingly, difficulties regulating negative emotions were also associated with increased FA in the left ATR. The left hemisphere has been primarily associated with the approach of positive stimuli, which will likely elicit positive emotions, while the right hemisphere is more involved in inhibiting actions that may lead to negative emotions. 51 As episodes of elated and depressed mood characterize BD, one might speculate that this lateralization might play a role in the switches between mood states. However, this is an unexpected finding that warrants replication. Additionally, future studies should explore in more detail how structural aberrations in primarily motivational networks affect emotion regulation.
Consistent with a previous study, 24 FA in the genu of the CC was a significant predictor of irritability. Consistent with previous studies, we observed elevated levels of irritability in BD youth 52 and to a lesser extent in REL. As outlined above, the anterior CC has been associated with working memory performance and sustained attention. 53 However, the hypothesis that impaired executive functions might mediate associations between aberrant WM microstructure in the anterior CC and irritability should be investigated in future studies.

| Limitations
We did not apply a cardiac-gated acquisition protocol. Thus, an impact of pulsatile motion artifacts on our results cannot be excluded, although the use of robust tensor fitting renders this unlikely. In addition, most patients were taking psychotropic medication and reported comorbid ADHD or/and anxiety disorders. Although additional analyses suggest that our results in these BD youth are not due to these confounding factors, small effects might have been undetected. Further, we included REL with diagnoses of anxiety and ADHD so as to not recruit an unusually resilient sample. This allowed to test, whether REL with and without diagnoses of ADHD and anxiety are comparable. Finally, a tractographic approach might have been more sensitive in the detecting differential age effects as TBSS projects the maximal value on the WM skeleton and might therefore fail to capture subtle structural abnormalities.

| CON CLUS ION
Our results show that aberrations in the microstructure of the CST and the body of the CC, both related to cognitive functions and aberrant emotion processing, are present not only in BD youth but also in relatives of individuals with BD suggesting that they might constitute a trait marker of BD. We also observed lower age-related FA increases in the anterior CC in BD youth only. Notably, FA in the anterior CC was negatively associated with levels of irritability, whereas FA in the ATR was associated with difficulties in emotion regulation, which we observed in BD youth and REL.

CO N FLI C T O F I NTE R E S T S
The author has no conflicts of interest.

AUTH O R CO NTR I B UTI O N
All authors have made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; and been involved in drafting the manuscript or revising it critically for important intellectual content; and given final approval of the version to be published. Each author agrees to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

S U PP O RTI N G I N FO R M ATI O N
Additional supporting information may be found online in the Supporting Information section.