*Copyright © 2013 Baribeau and Anagnostou. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# **Upregulated GABA inhibitory function in ADHD children with child behavior checklist–dysregulation profile: 123I-iomazenil SPECT study**

*Shinichiro Nagamitsu1 \*, Yushiro Yamashita1 , Hitoshi Tanigawa2 , Hiromi Chiba3 , Hayato Kaida2 , Masatoshi Ishibashi 2 , Tatsuyuki Kakuma4 , Paul E. Croarkin5 and Toyojiro Matsuishi 1*

*1 Department of Pediatrics and Child Health, Kurume University School of Medicine, Fukuoka, Japan, 2 Department of Radiology, Kurume University School of Medicine, Fukuoka, Japan, 3 Department of Psychiatry, Kurume University School of Medicine, Fukuoka, Japan, 4 Biostatistics Center, Kurume University School of Medicine, Fukuoka, Japan, 5 Department of Psychiatry and Psychology, Mayo Clinic, Rochester, MN, USA*

#### *Edited by:*

*Stephanie Ameis, The Hospital for Sick Children and University of Toronto, Canada*

#### *Reviewed by:*

*Rosana Lima Pagano, Hospital Sírio-Libanês, Brazil Mehereen Bhaijiwala, The Hospital for Sick Children, Canada*

#### *\*Correspondence:*

*Shinichiro Nagamitsu, Department of Pediatrics and Child Health, Kurume University School of Medicine, 67 Asahi-machi Kurume City, Fukuoka 830-0011, Japan<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neuropsychiatric Imaging and Stimulation, a section of the journal Frontiers in Psychiatry*

> *Received: 16 February 2015 Accepted: 18 May 2015 Published: 02 June 2015*

#### *Citation:*

*Nagamitsu S, Yamashita Y, Tanigawa H, Chiba H, Kaida H, Ishibashi M, Kakuma T, Croarkin PE and Matsuishi T (2015) Upregulated GABA inhibitory function in ADHD children with child behavior checklist–dysregulation profile: 123I-iomazenil SPECT study. Front. Psychiatry 6:84. doi: 10.3389/fpsyt.2015.00084* The child behavior checklist–dysregulation profile (CBCL–DP) refers to a pattern of elevated scores on the attention problems, aggression, and anxiety/depression subscales of the child behavior checklist. The aim of the present study was to investigate the potential role of GABA inhibitory neurons in children with attention deficit/hyperactivity disorder (ADHD) and dysregulation assessed with a dimensional measure. Brain single photon emission computed tomography (SPECT) was performed in 35 children with ADHD using 123I-iomazenil, which binds with high affinity to benzodiazepine receptors. Iomazenil binding activities were assessed with respect to the presence or absence of a threshold CBCL–DP (a score *≥*210 for the sum of the three subscales: Attention Problems, Aggression, and Anxiety/Depression). We then attempted to identify which CBCL–DP subscale explained the most variance with respect to SPECT data, using "age," "sex," and "history of maltreatment" as covariates. Significantly higher iomazenil binding activity was seen in the posterior cingulate cortex (PCC) of ADHD children with a significant CBCL–DP. The Anxiety/Depression subscale on the CBCL had significant effects on higher iomazenil binding activity in the left superior frontal, middle frontal, and temporal regions, as well as in the PCC. The present brain SPECT findings suggest that GABAergic inhibitory neurons may play an important role in the neurobiology of the CBCL–DP, in children with ADHD.

**Keywords: CBCL-dysregulation profile, iomazenil, GABA, ADHD**

## **Introduction**

Severe behavioral and affective dysregulation with symptoms, such as hyperactivity, aggression, irritability, mood instability, and anxiety, contribute to significant academic and psychosocial impairment in children. Some of these symptoms are consistent with attention deficit hyperactivity disorder (ADHD). ADHD is the most frequent neuropsychiatric disorder in children and often presents with co-occurring disruptive behavior disorders, anxiety disorders, and bipolar disorder. Hyperactivity, irritability, and impulsivity place children at risk of maltreatment as a result of strained parent–child interactions (1, 2). The insecure parent–child relationship further exacerbates the behavioral and affective dysregulation observed in children.

The child behavior checklist-dysregulation profile (CBCL–DP) refers to a pattern of elevated scores on the Attention Problems, Aggression, and Anxiety/Depression subscales of the child behavior checklist (CBCL) (3). The CBCL–DP was originally proposed as a means of identifying youth with bipolar disorder (4). However, recent studies suggest that the results of the CBCL–DP are not simply an early manifestation of a single disease process, but rather that the CBCL–DP can be used as a developmental risk marker for a persisting deficit in self-regulation of affect and behavior (5, 6). The CBCL–DP may be best interpreted as an indicator of symptom severity and functional impairment (7, 8). Children with ADHD who had a threshold level CBCL–DP score (*≥*210) showed higher rates of comorbidity disorders, including oppositional defiant disorder (ODD), conduct disorder (CD), anxiety disorder, bipolar disorder, and depression (9). The CBCL–DP is also associated with mood, anxiety, disruptive behavior disorders, and substance use in adulthood (3).

The underlying neurobiological defects or aberrant neuronal activity leading to the dysregulation profile in children with ADHD are elusive. Reducing serotoninergic function in children with ADHD and a significant CBCL–DP resulted in slower cognitive performance compared to children with ADHD who did not have the CBCL–DP, indicating that serotoninergic function could play a decisive role in the etiology of the CBCL–DP (10). In addition, the CBCL subscale of "Aggression" was found to be the main discriminator of ADHD children with CBCL–DP versus those without CBCL–DP with respect to serotoninergic dysfunction. Conversely, prior translational work with magnetic resonance spectroscopy and transcanial magnetic stimulation paradigms suggest that GABAergic neurochemistry and neurotransmission are dysregulated in children with ADHD (11). Ongoing work also suggests that defects in the GABAergic system in adults increase an individual's vulnerability to severe psychiatric illnesses due to aberrant regulation of serotoninergic and/or dopaminergic neurons (12, 13). Previous biochemical and pharmacological studies indicate that deficits in GABA receptor function, induced by intravenous infusion of iomazenil followed by a serotoninergic agonist, predispose healthy volunteers to increased anxiety and dissociative disturbances, suggesting that deficits in the GABAergic system may contribute to the pathophysiology of serotonininduced psychosis (12).

123I-iomazenil is a radioactive ligand for central-type benzodiazepine receptors that forms a complex with GABA(A) receptors. Thus, 123I-iomazenil single photon emission computed tomography (SPECT) can indirectly index GABA receptor function. 123Iiomazenil is a frequently used radionuclide tracer for presurgical evaluation of patients with refractory partial epilepsy (14, 15). Moreover, recent neuroimaging studies have explored the role of GABAergic inhibitory function in psychiatric disorders such as schizophrenia, anxiety disorders, and developmental disorders (16–20). To our knowledge, there is no previous work which characterizes GABA receptor functioning with 123I-iomazenil SPECT among children with ADHD. The working hypothesis of the present study was that behavioral and affective symptoms in children with ADHD, reflected in CBCL–DP scores, would correlate with changes in cortical GABAergic neuronal activity. To confirm this hypothesis, brain SPECT was performed using 123Iiomazenil in ADHD children with or without CBCL–DP. Further, we tried to identify which of the three significant scales in the CBCL–DP explains the most variance with respect to SPECT data using "age," "sex," and "history of maltreatment" as covariates.

### **Materials and Methods**

### **Ethics Statement**

The design of the study and procedures for obtaining informed consent were approved by the Medical Ethics Committee of Kurume University School of Medicine (#10081). Informed consent was obtained from each child and his/her parents prior to their participation in the study.

### **Participants**

Thirty-five children with ADHD (23 boys, 12 girls) enrolled in the study. Participants were recruited after visits from the Department of Pediatrics, Kurume University, for the management of externalizing symptoms (e.g., difficulty maintaining attention, restlessness, hyperactivity, and aggressive behavior) or internalizing symptoms (e.g., anxiety, dissociation, and depressive symptoms). A diagnosis of ADHD was made using the *Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, Text Revision (Dsm-Iv-Tr)* (21). Children who had anxious or depressive symptoms, but did not have ADHD symptoms were excluded in this study. Of the 35 child participants with ADHD, 15 had the combined type, 11 had hyperactive-impulsive type, and 9 had inattentive type. Seventeen children (7 male, 10 females) had experienced an obvious maltreatment, such as physical (*n* = 9), psychological (*n* = 6), or sexual abuse (*n* = 1), or sexual assault (*n* = 1) during preschool. In 11 of these instances, a child-welfare consultation center had previously supported the families in hopes of preventing maltreatment. Two children stayed in a child residential care institution. However, none of the participants met the diagnostic criteria for posttraumatic stress disorder (PTSD) on assessment. Seven of the 35 subjects (20%) had comorbid disorders, such as ODD (*n* = 2), CD (*n* = 1), anxiety disorder (*n* = 2), and depression (*n* = 2). The mean age of the children at the time of their hospital visit was 10.4 years. All participants were medication naïve prior to enrollment.

### **Child Behavior Checklist**

Behavioral and psychiatric assessments of the children included the CBCL, ADHD rating scale (hyperactivity/impulsive and inattention scores, as well as total score) (22, 23), the Child Depression Inventory (CDI), the Child Dissociative Checklist (CDC), and the Wechsler Intelligence Scale for Children (WISC-III). The CBCL was used to evaluate children's emotional and behavioral functioning, competencies, and social problems, with specific items evaluating internalizing and externalizing symptoms, as well as attention and thought problems. Items evaluating internalizing symptoms focus on withdrawal, somatic complaints, and anxiety/depression. Items evaluating externalizing symptoms focus on delinquent or aggressive behavior. The CBCL–DP refers to Nagamitsu et al. GABA in children with ADHD

a pattern of elevated scores on the Attention Problems, Aggression, and Anxiety/Depression subscales of the CBCL. A threshold CBCL–DP was defined as a score *≥*210 for the sum of three subscales. (4) Physicians rated the participants using the ADHD rating scale, CDI, and CDC, and the parents rated their children using the CBCL.

### **Iomazenil Single Photon Emission Computed Tomography and Analysis of Regions of Interest**

All 35 children underwent iomazenil SPECT imaging of the brain. Briefly, children were injected intravenously with a bolus of 95–117 MBq 123I-iomazenil (Nihon Medi-Physics, Tokyo, Japan), which binds with high affinity to benzodiazepine receptors. The SPECT scan was performed 3 h after injection of the tracer, without any sedation, using a large field-of-view dual-detector camera and a computer system equipped with a low-energy, high-resolution, parallel-hole collimator. The dual detector camera rotated over 180° in a circular orbit and in 32 steps of 40 s each to cover 360° in about 22 min. Brain magnetic resonance imaging (MRI) was performed using a superconducting magnet operating at 1.5 T. For coregistered SPECT and MRI analysis, a method of image integration was applied using Fusion Viewer software (Nihon Medi-Physics) with a registration algorithm based on maximum mutual information (**Figure 1**). Subsequently, the cortical and subcortical regions of interest (ROIs) in the acquired SPECT data were defined. Using elliptical templates, the ROIs were placed over the following regions: the superior frontal, middle frontal, parietal, temporal, and occipital regions in each hemisphere; the midbrain; and the anterior and posterior cingulate cortex (ACC and PCC, respectively; **Figure 1**). Each relative iomazenil binding activity in ROIs was expressed as a ratio of that in the occipital cortex. As 123I-iomazenil affinity in the occipital region was maximum and stable in brain cortex, the occipital region was used as a reference (24).

### **Data Analysis**

The differences of each CBCL subscale, ADHD-RS, CDI score, CDC score, and Intelligence scale between ADHD children with/without CBCL–DP were compared by student's *t*-test. We first analyzed correlations between the relative iomazenil binding activity expressed as a ratio in each ROI and psychometric profiles after controlling for the effects of age, sex, and history of maltreatment. Further, we compared iomazenil binding activity with respect to the presence or absence of a threshold CBCL–DP score in these children and tried to identify which of the three CBCL–DP subscales explained the most variance with respect to the SPECT data. Associations between each of the CBCL–DP subscales and iomazenil binding activity in each brain area were evaluated using liner regression models, with "age," "sex," and "history of maltreatment" as covariates.

## **Results**

Behavioral and psychiatric assessments of the participants were shown in **Table 1**. Of the 35 participants, 15 had a threshold CBCL–DP score (i.e., a score *≥*210) and 20 had CBCL–DP scores *<*210. The group with threshold CBCL–DP scores had a lower

**FIGURE 1 | Designated regions of interest (ROIs) in fusion images of 123I-iomazenil SPECT and MRI**. The top panel shows brain MRI (transverse and sagittal T1 sequences), the middle panel shows corresponding results of 123I-iomazenil SPECT, and the bottom panel shows fusion imaging. Outlined regions in the bottom panel indicate designated ROIs, namely (a) the superior frontal, (b) parietal, (c) middle frontal, (d) temporal, (e) occipital regions, (f) anterior, and (g) posterior cingulate gyrus.

ratio of male to female participants and more instances of maltreatment. Each ADHD rating scale, all subscales of CBCL with the exception of somatic problems, and CDC score were significantly higher in ADHD children with threshold CBCL–DP scores. Four participants with threshold level CBCL–DP scores had comorbidity disorders, including depression (*n* = 2), ODD (*n* = 1), and CD (*n* = 1). Three participants without threshold CBCL–DP scores had comorbidity disorders, including anxiety disorder (*n* = 2) and ODD (*n* = 1). There was a difference in the CBCL–DP scores between participants with/without comorbidity disorders; however, this difference did not reach statistical significance (*n* = 28, 199 *±* 20, and *n* = 7, 209 *±* 12, respectively, *p* = 0.10).

Analyses of all participants (*n* = 35) revealed correlations between iomazenil binding activity in several brain regions and some part of the CBCL profile, after controlling for the effects of age, sex, and a history of maltreatment (**Table 2**). In both ACC and PCC, iomazenil binding activity had a statistically significant positive correlation with scores on the Anxiety/Depressed (**Table 2**; **Figure 2**), Internalizing, and Withdrawal Problems subscales of the CBCL (**Table 2**). In addition, significant positive correlations were noted for iomazenil binding activity in the ACC and Thought Problems on the CBCL, as well as for iomazenil binding activity in the PCC and Attention problems and Social Problems on the CBCL (**Table 2**). These significant correlations were not seen for other combinations in other brain regions, except for iomazenil binding activity in the midbrain and Thought Problems on the CBCL, and iomazenil binding activity in the right temporal region and Social Problems on the CBCL. There were no significant correlations between iomazenil binding activities in any brain region and any of the ADHD rating scales, CDI score, and CDC score

*CBCL–DP, child behavior checklist–dysregulation profile; ADHD-RS, attention deficit hyperactivity disorder rating scale; CDI, child depression inventor; CDC, children dissociative checklist; WISC, Wechsler intelligence scale for children.*

*Significant difference from children without significant CBCL–DP (\*indicates p < 0.05, \*\*indicates p < 0.001).*

(data not shown). Iomazenil binding activity in the PCC was significantly higher in ADHD children with a threshold CBCL–DP score than in ADHD children with scores *<*210 after controlling for the effects of age, sex, and a history of maltreatment (**Table 3**, *F*-value = 4.36, *p <* 0.05). Of the three CBCL–DP subscales, the Anxiety/Depression subscale had significant effects on higher iomazenil binding activity in the left superior frontal, middle frontal, and temporal regions, as well as in the PCC (**Table 4**).

## **Discussion**

This is the first neuroimaging study showing that behavioral and affective symptoms in children with ADHD, reflected in CBCL–DP scores, are correlated with changes in cortical GABAergic neuronal activity. Overall, increased iomazenil activity in the ACC and PCC was associated with higher scores on many of the CBCL subscales. In ADHD children with a significant CBCL–DP, iomazenil activity was upregulated in the PCC. Of the three CBCL–DP subscales, the Anxiety/Depression subscale had a significant effect on iomazenil binding activity in many brain regions. These results suggest that behavioral and affective dysregulation in ADHD children may be characterized by changes of GABAergic neural activity. In this section, we discuss the role of the cingulate cortex in GABA function, the association between CBCL–DP scores and GABA function, and age-dependent differences in GABA function.

The cingulate cortex is one of the largest parts of the limbic lobe and the prefronto-limbic circuitry. The ACC plays key roles in emotion, motivation, and motor functions, whereas the PCC is involved in emotion, facial recognition, and memory functions (25–27). In the present study, we found higher iomazenil binding activity in ACC and PCC that was associated with higher scores on many of the CBCL subscales in ADHD children with and without CBCL–DP. Similar findings have been reported in healthy adults. For example, Kim et al. (28) found a positive correlation in healthy subjects between high GABA concentrations in the ACC and a high harm avoidance temperament, characterized by worrying about potential problems, fearful of uncertainties, and being shy in unfamiliar environments. Moreover, increased activity in the PCC has been observed in emotional disorders, including obsessive–compulsive disorder, major depression, and social phobia (29, 30). Because the cingulate cortex has been suggested to have an important role in modulating human fear and anxiety by modulating the activity of other limbic structures, including the amygdala (31), the increased GABAergic function in the cingulate cortex of ADHD children in the present study may have inhibited excessive excitation of the limbic system, which contributes to the development of behavioral and affective dysregulation.

We found that the Anxiety/Depression subscale of the CBCL–DP explains the most variance with respect to SPECT data in various brain regions using "age," "sex," and "history of maltreatment" as covariates. The Aggression and Attention Problem subscales of the CBCL–DP had no significant effects on SPECT data in various brain regions. These findings strongly support previous converging lines of evidence regarding the association between GABAergic activation and increased anxiety (32). Conversely, several biochemical and genetic studies have provided evidence of a significant role of serotoninergic function in aggressive behavior. For example, an inverse correlation has been reported between downregulated platelet or CSF 5-hydroxyindolecetic acid (5-HIAA), a major metabolite of serotonin, and levels of aggression and impulsivity (33, 34). Furthermore, Haberstick et al. (35) reported an association between certain promoter polymorphisms in the serotonin transporter (5HTTLPR) and greater aggressive behavior in middle childhood, suggesting that differences in serotonergic functioning may be a contributing factor to different levels of aggressive behavior. In terms of the biological mechanism underlying attention function, an important role for dopaminergic neurons has been proposed. Several neuroimaging studies have shown aberrant dopamine transporter (DAT) levels in the nucleus accumbens, caudate, and midbrain, as well as a positive relationship between DAT levels in the putamen and inattention scores in ADHD patients (36–38). Together, these findings suggest that changes in several neurotransmitter systems, including serotoninergic, dopaminergic, and GABAergic neurons, are likely to be involved in constructing the clinical manifestations of the CBCL–DP.

Significant positive correlations between GABAergic inhibitory function and the Anxiety/Depression subscale were also seen in our study. Although previous neuroimaging studies have reported those correlations in adulthood with psychiatric disorders (17, 28), the present study is the first report of the correlation in childhood with psychiatric disorders. Despite the positive correlation in childhood, previous neuroimaging studies using iomazenil SPECT have revealed negative correlations between

#### **TABLE 2 | Partial correlation coefficients between CBCL profiles and the iomazenil binding activites in each brain region**.

*CBCL, child behavior checklist; ACC, anterior cingulate cortex; PCC, posterior cingulate cortex; R, right; L, left.*

*\*Indicates p < 0.05; \*\*indicates p < 0.01.*

*a Indicates subscale which comprises CBCL–DP (dysregulation profile).*

**TABLE 3 | Effect of the significant CBCL–DP (score** *≥***210) on iomazenil binding activities in each brain region**.

*CBCL–DP, child behavior checklist–dysregulation profile.*

GABA–benzodiazepine receptor binding activity and the severity of anxiety symptoms in adults with panic or traumatic disorders (17–19). It is well known that there are considerable changes in the number of GABA receptors and in subunit expression during brain development (39). Specifically, the greatest number of GABA receptors is found in the youngest children, with numbers decreasing exponentially with age, and there are age-related increases in α1-subunit-containing GABA receptors (40). These age-related changes in GABA receptors may affect outcomes when assessing increases and/or decreases in overall iomazenil binding activity in children.

The present study has several limitations that require consideration in future studies. For example, in the present study, SPECT exhibited poor resolution around some limbic regions, such as the amygdala and hippocampus, which are important for emotion processing. In these small regions, the obtained radioactivity might differ from the true activity because of partial volume effect (PVE). The PVE can be defined as the underestimation of binding per unit brain volume in small objects or regions because of the blurring of the radioactivity (spillout and spill-in) between regions. These regions need to be resolved using MR imaging-based correction for PVE (41, 42). Brain imaging data from normal healthy children are not available

*CBCL–DP, child behavior checklist–dysregulation profile.*

*\*Indicates significant effects on higher iomazenil binding activity.*

because of ethical concerns with SPECT studies of this population. Therefore, we focused our research questions on the correlation between GABAergic inhibitory function in specific brain regions and psychometric profiles. It is possible that, in addition to the

### **References**

population of people with ADHD, our result is generalizable to the normal population. Furthermore, we selected iomazenil activity in the occipital regions as a reference, meaning that we could not evaluate inhibitory function in occipital regions. We did not clarify how putative dopaminergic or serotoninergic changes are involved in other subscales, such as the Aggression and Attention Problem subscales, of the CBCL–DP. It is possible that investigations incorporating the simultaneous assessment of benzodiazepine receptor binding activity and homovanillic acid (HVA) and 5HIAA in the urine (principal metabolites of dopamine and serotonin, respectively) could provide new insights into the underlying neurobiological defects or aberrant neuronal activity leading to the dysregulation profile in children.

In conclusion, the present 123I-iomazenil brain SPECT study provides evidence that changes in GABAergic inhibitory neuronal activity correlate with some elements of function measured by the CBCL–DP. Brain SPECT may be useful for the evaluation of the possible pathogenesis of neuropsychiatric symptoms observed in children.

### **Author Contributions**

SN participated in the design of this study and compiled the manuscript. SN, YY, and HC saw the patients and obtained informed consent and their agreement to participate in the study. Diagnosis of comorbidity disorders was made by HC. SN and HC summarized participant behavioral and psychiatric assessments, including CBCL–DP data. Three radiologists (HT, HK, and MI) were in charge of radioactive measurements and calculations of iomazenil activity using ROIs. TK, a statistician, conducted the statistical analyses. PC and TM supervised the preparation of the manuscript.

### **Acknowledgments**

This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology (#22591143, #25460643).

suicidality, and functional impairment: a longitudinal analysis. *J Child Psychol Psychiatry* (2011) **52**:139–47. doi:10.1111/j.1469-7610.2010.02309.x

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Nagamitsu, Yamashita, Tanigawa, Chiba, Kaida, Ishibashi, Kakuma, Croarkin and Matsuishi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# A preliminary study of white matter in adolescent depression: relationships with illness severity, anhedonia, and irritability

#### **Sarah E. Henderson1 , Amy R. Johnson1 , Ana I. Vallejo1 , Lev Katz 1 , EdmundWong1 and Vilma Gabbay 1,2\***

1 Department of Psychiatry, Icahn School of Medicine at Mount Sinai, New York, NY, USA 2 Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY, USA

#### **Edited by:**

Paul Croarkin, Mayo Clinic, USA

#### **Reviewed by:**

Faisal Al-Otaibi, Alfaisal University, Saudi Arabia Niels Bergsland, Buffalo Neuroimaging Analysis Center, USA Kirti Saxena, Baylor College of Medicine, USA Susannah J. Tye, Mayo Clinic, USA

#### **\*Correspondence:**

Vilma Gabbay, Department of Psychiatry, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA e-mail<EMAIL_ADDRESS>
Major depressive disorder (MDD) during adolescence is a common and disabling psychiatric condition; yet, little is known about its neurobiological underpinning. Evidence indicates that MDD in adults involves alterations in white and gray matter; however, sparse research has focused on adolescent MDD. Similarly, little research has accounted for the wide variability of symptom severity among depressed teens. Here, we aimed to investigate white matter (WM) microstructure between 17 adolescents with MDD and 16 matched healthy controls (HC) using diffusion tensor imaging. We further assessed within the MDD group relationships between WM integrity and depression severity, as well as anhedonia and irritability – two core symptoms of adolescent MDD. As expected, adolescents with MDD manifested decreased WM integrity compared to HC in the anterior cingulum and anterior corona radiata. Within the MDD group, greater depression severity was correlated with reduced WM integrity in the genu of corpus callosum, anterior thalamic radiation, anterior cingulum, and sagittal stratum. However, anhedonia and irritability were associated with alterations in distinctWM tracts. Specifically, anhedonia was associated with disturbances in tracts related to reward processing, including the anterior limb of the internal capsule and projection fibers to the orbitofrontal cortex. Irritability was associated with decreased integrity in the sagittal stratum, anterior corona radiata, and tracts leading to prefrontal and temporal cortices. Overall, these preliminary findings provide further support for the hypotheses that there is a disconnect between prefrontal and limbic emotional regions in depression, and that specific clinical symptoms involve distinct alterations in WM tracts.

**Keywords: depression, adolescent, white matter, diffusion tensor imaging, anhedonia, irritability**

### **INTRODUCTION**

Major depressive disorder (MDD) in adolescence is a prevalent and disabling psychiatric illness associated with serious consequences including academic failure, social withdrawal, substance abuse, and most critically, suicide (1–5). Converging evidence derived from neuroimaging studies suggests that adolescent MDD entails morphological, functional, and neurochemical alterations (6–10). Importantly, since adolescence represents a critical period of rapid neuroplasticity [e.g., increased myelination, synaptic pruning; (11–13)], white matter (WM) alterations can contribute to the neurobiology of adolescent MDD. Indeed, in our prior investigation of gamma-Aminobutyric acid in the anterior cingulate cortex (ACC), we incidentally found reduced WM volume in adolescents withMDD compared to healthy controls [HC; (9)]. However, there has been sparse research focusing on WM alterations in this age group.

Diffusion tensor imaging (DTI) enables the non-invasive examination of *in vivo* structural connectivity by providing measures of WM microstructure and integrity based on the extent of water diffusion (14). Several DTI measures are typically quantified, with fractional anisotropy (FA) being the most commonly used

to reflect WM integrity. Higher FA values suggest greater diffusion in the direction of the axon, and thus greater WM integrity. Other measures, including mean diffusivity (MD), radial diffusivity (RD), and axial diffusivity (AD), can also be determined to investigate different aspects of WM microstructure.

To date, most DTI research in MDD has investigated adults and consistently reported decreased FA in tracts connected to the prefrontal cortex (PFC) or tracts connecting the two hemispheres within the PFC (15, 16). Only one DTI study was carried out in adolescents with depression, demonstrating a similar pattern to adult MDD of reduced FA in tracts connected to the subgenual ACC and the PFC [i.e., uncinate fasciculus, inferiorfronto-occipital fasciculus, anterior cingulum, superior longitudinal fasciculus; (17)]. However, results may have been impacted by the concurrent use of psychotropic medication and past substance abuse in some subjects. Relatedly, medication-naïve adolescents with a familial risk for unipolar depression also demonstrated reduced FA compared to HC in similar tracts (18).

In this study, we aimed to expand on prior work by examining WM integrity in psychotropic medication-free adolescents with MDD compared to HC. Given the inherent heterogeneity of adolescent MDD, we further sought to identify specific WM alterations in relation to MDD severity as well as anhedonia and irritability – two core symptoms of adolescent MDD. Due to our desire to explore a range in depression severity, we included patients with mild to moderate severity. Based on others' and our prior findings (8, 15–17), we hypothesized that adolescents with MDD would manifest less restricted diffusion (i.e., decreased WM integrity) compared to HC in tracts connecting frontal, striatal, and limbic regions. We also predicted that similar tracts would be associated with depression severity. For anhedonia, we expected reduced WM integrity in tracts that have been implicated in reward-related processing in the ventral striatum [i.e., subgenual cingulate, forceps minor, inferior-fronto-occipital fasciculus, anterior thalamic radiation (ATR), anterior limb of the internal capsule; (19)], and that these would be distinct from those associated with irritability. Finally, only one study has examined irritability in a clinical population [i.e., Huntington's disease; (20)] and found involvement of the amygdala. As such, we predicted the impairment of tracts connecting to the amygdala (e.g., uncinate fasciculus, inferior-fronto-occipital fasciculus, inferior longitudinal fasciculus).

### **MATERIALS AND METHODS**

#### **PARTICIPANTS**

The sample population partially overlapped with that used for our previously published resting state functional magnetic resonance imaging research (8), and was comprised of 17 adolescents with MDD (ages 13–20 years, *M* = 16.8, *SD* = 2.2, 8 female) and 16 HC (ages 13–19 years, *M* = 16.4, *SD* = 1.4, 10 female) group matched for age, and all were right-handed. Depressed adolescents were recruited through the New York University (NYU) Child Study Center, the Bellevue Hospital Center Department of Psychiatry, and local advertisements in the NY metropolitan area. HC were recruited from the greater NY metropolitan area through local advertisements and from the families of NYU staff. The study was approved by the NYU School of Medicine and the Icahn School of Medicine at Mount Sinai institutional review boards. Prior to enrollment, study procedures were explained to the subjects and parents. Written informed consent was provided by participants age 18 and older; those under age 18 provided signed assent and a parent/guardian provided signed informed consent.

#### **Inclusion and exclusion criteria**

All subjects were≤20 years old and did not present with any significant medical or neurological disorders. Other exclusionary criteria consisted of an IQ < 80, MRI contraindications as assessed by a standard screening form, a positive urine toxicology test or a positive urine pregnancy test in females.

All MDD subjects met the DSM-IV-TR diagnosis of MDD with a current episode ≥8 weeks duration, and raw severity score ≥40 (i.e., T score ≥ 63) during the initial evaluation on the Children's Depression Rating Scale-Revised (CDRS-R), and were psychotropic medication-free for at least 7 half-lives of the medication. To explore a wider range of depression severity we included patients presenting with mild to severe depression on the date of the scan. Exclusionary criteria for the MDD group included current or past DSM-IV-TR diagnoses of bipolar disorder, schizophrenia, pervasive developmental disorder, panic disorder, obsessive-compulsive disorder, conduct disorder, Tourette's disorder, or a substance-related disorder in the past 12 months. Current diagnoses of post-traumatic stress disorder or an eating disorder were also exclusionary. In addition, acute suicidality requiring immediate inpatient admission was exclusionary. HC subjects did not meet the criteria for any major current or past DSM-IV-TR diagnoses and had never received psychotropic medication.

#### **CLINICAL ASSESSMENTS**

All subjects were assessed by a board-certified child and adolescent psychiatrist or a clinical psychologist at the NYU Child Study Center. Clinical diagnoses were established using the Schedule for Affective Disorders and Schizophrenia for School-Age Children-Present and LifetimeVersion [KSADS-PL; (21)], a semi-structured interview performed with both the subjects and their parents. Depression severity was assessed by the CDRS-R and the Beck Depression Inventory, second edition [BDI-II; (22)]. Additionally, suicidality and anxiety were assessed using the Beck Scale for Suicidal Ideation [BSSI; (23)] and the Multidimensional Anxiety Scale for Children [MASC; (24)], respectively. The Kaufman Brief Intelligence Test (25) or the Wechsler Abbreviated Scale of Intelligence (26) were used to estimate IQ. Urine toxicology and pregnancy tests were administered on the day of the scan.

#### **Anhedonia**

Our approach to quantifying anhedonia allows for clinician- and self-rated assessments to contribute equally to the anhedonia score (range 1–13). As in our previous studies (8, 9, 27), the score for each subject was computed by summing the responses to three items associated with anhedonia from the clinician-rated CDRS-R (item 2: "difficulty having fun;" scale of 1–7) and the self-rated BDI-II (items 4: "loss of pleasure" and 12: "loss of interest;" scales of 0–3).

#### **Irritability**

Our approach again combined both clinician- and self-rated assessments to contribute to the irritability score (range 1–10). The score for each subject was computed by summing the responses to the items associated with irritability from the CDRS-R (item 8: "irritability;" scale of 1–7) and the BDI-II (item 17: "irritability;" scale of 0–3).

#### **DATA ACQUISITION AND ANALYSIS**

Diffusion data were acquired on a Siemens Allegra 3.0 T scanner at the NYU Center for Brain Imaging using a single-channel head coil. Diffusion-weighted echo-planar images (EPI) were acquired along 12 diffusion gradient directions for acquisition of 35 slices through the whole brain (TR = 6000 ms, TE = 82 ms, flip angle = 90°,*b* value = 1000 s/mm2 , FOV = 192 mm,128 × 128 matrix, slice thickness = 2.5 mm, with four averages). Highresolution T1-weighted anatomical images were acquired using a magnetization-prepared gradient echo sequence (TR = 2530 ms; TE = 3.25 ms; TI = 1100 ms; flip angle = 7°; 128 slices; FOV = 256 mm; acquisition voxel size = 1.3 mm × 1 mm × 1.3 mm).

All preprocessing was performed using FMRIB's Software Library (FSL; Oxford, UK): FMRIB's Diffusion Toolbox (FDT). Preprocessing began with eddy current correction to correct for gradient-coil distortions and small head motions, using affine registration to a b0 reference volume. A diffusion tensor model was fitted to each voxel along the principal λ1, λ2, λ3 directions to generate FA, MD, RD, and AD. We then implemented FSL's tract-based spatial statistics pipeline [TBSS; (28)], in which a nonlinear registration aligned each subject to the FMRIB58\_FA template in 1 mm × 1 mm × 1 mm standard space, and then warped each subject into standard Montreal Neurological Institute space (MNI152). A WM "skeleton" was then generated representing a single line running down the centers of all of the common WM fibers by using an FA cut-off of 0.2, and relevant diffusivity measures (i.e., FA, MD, RD, AD) were projected onto the skeleton. Group statistical analysis was then conducted only on voxels within the WM skeleton mask, therefore restricting the voxel-wise analysis only to voxels with high confidence of lying within equivalent major WM pathways in each individual.

In order to assess differences in FA, MD, RD, and AD between the MDD and HC groups, we used FSL's Randomise with 5000 permutations to perform voxel-wise independent samples *t*-tests using voxel-based thresholding while controlling for age and sex. Group comparisons did not withstand stringent correction for multiple comparisons using family-wise error correction (FWE; implemented by Randomise) or FDR correction (implemented by FSL's FDR program). As such, we performed a second, more exploratory analysis in which we accepted clusters of at least 10 contiguous voxels at *p* < 0.001.

To investigate relationships with depression severity, anhedonia, and irritability, Randomise was again used to perform a series of one-sample *t*-tests using the scorefor each category, respectively, while controlling for age and gender. Due to the low variability in scores in the HC group, as well as the nature of the study topic, the analysis was limited to the MDD group whose scores were normally distributed. Once again, tests did not withstand correction for multiple comparisons and we used the more exploratory approach of accepting clusters of at least 10 contiguous voxels at *p* < 0.001. We used FSL's cluster program to extract all clusters across the brain, and anatomical localization of each cluster was determined using the FSLView atlas toolbox and the relevant gray matter (Harvard-Oxford Cortical/Subcortical) and WM (Johns Hopkins UniversityWM tractography) atlases. Brain imaging results were prepared for display using FSL's tbss\_fill script, which displays results superimposed upon the WM skeleton from the group TBSS analysis.

#### **RESULTS**

#### **PARTICIPANTS**

Demographic and clinical characteristics are summarized in **Table 1**. One subject with MDD had been treated with Lexapro and Ambien for 7 months, but was medication-free for approximately 14 months prior to scanning. A second subject with MDD had a brief trial with Prozac which was self-discontinued prior to participation in this study. All other subjects were psychotropic medication-naïve. Fifteen subjects with MDD had experienced **Table 1 | Demographic and clinical characteristics of adolescents with major depressive disorder (MDD) and healthy controls.**

aRespective percentages (may not add up to 100% due to rounding).

bChildren's depression rating scale – revised.

cBeck depression inventory, 2nd ed.

dBeck scale for suicidal ideation.

eMultidimensional anxiety scale for children.

fAttention deficit hyperactivity disorder.

gGeneralized anxiety disorder.

only one episode of depression, with length of episode ranging from 4 to 48 months, and two patients reported having two distinct episodes. Two Shapiro–Wilk tests revealed that anhedonia and irritability were both normally distributed within the MDD group, *p*s = 0.81, 0.48, respectively. Depression severity (excluding the anhedonia- or irritability-related items) was significantly correlated with both severity of anhedonia (*r* = 0.66, *p* < 0.005) and irritability (*r* = 0.66, *p* < 0.005) within our MDD sample. Anhedonia and irritability were not correlated (*r* = 0.37, *p* = 0.15).

#### **WHOLE-BRAIN GROUP COMPARISON**

No voxel-wise group comparisons for FA, MD, RD, or AD withstood correction for multiple comparisons. An exploratory analysis using a threshold of *p* < 0.001, uncorrected with clusters exceeding 10 contiguous voxels, revealed 4 significant clusters (**Table 2**, **Figure 1**). Compared with HC, the MDD group had lower FA in the anterior cingulum, and lower AD in the anterior corona radiata (ACR). However, the MDD group also had greater FA and lower RD in the posterior cingulum compared to HC.

#### **Table 2 | Voxel-wise group comparison results.**

Units for AD and RD = mm2 /s. Coordinates in MNI space. Threshold p < 0.001, uncorrected, k > 10. COG, center of gravity; FA, fractional anisotropy; MD, mean diffusivity; RD, radial diffusivity; AD, axial diffusivity; WM, white matter; L, left; R, right; ACR, anterior corona radiata.

**FIGURE 1 | (A)** Increased WM integrity in the MDD group vs. HC in the posterior cingulum near the hippocampus; **(B)** decreased WM integrity in the MDD group vs. HC in the anterior cingulum near the precuneus.

#### **WHOLE-BRAIN CORRELATIONS WITH DEPRESSION SEVERITY IN MDD**

Exploratory analyses revealed a total of 16 uncorrected clusters, with 5 overlapping clusters between the 4 diffusivity measures (**Table 3**, **Figure 2**). As depression severity increased, FA decreased in the genu of the corpus callosum, the sagittal stratum, the ATR, and the anterior cingulum. Additionally, a positive correlation between MD and depression severity was found in the same sagittal stratum cluster as the FA analysis, as well as in clusters in the ATR and corticospinal tract. Similarly, illness severity was positively correlated with RD in the same sagittal stratum cluster as the FA and MD analyses, the same genu of the corpus callosum and

ATR clusters as the FA analysis, the same ATR cluster as the MD analysis, and a cluster in the superior longitudinal fasciculus (SLF). Finally, increased illness severity was associated with increased AD in the same corticospinal cluster as the MD analysis as well as in clusters in the inferior-fronto-occipital fasciculus (IFOF), the SLF, and fibers projecting to the orbitofrontal cortex (OFC).

#### **WHOLE-BRAIN CORRELATIONS WITH ANHEDONIA IN MDD**

Analyses revealed a total of 14 uncorrected clusters, with 2 overlapping clusters between FA and RD (**Table 4**,**Figure 3**). As anhedonia increased, FA increased in a cluster in the posterior cingulum near

#### **Table 3 | Voxel-wise correlations with depression severity (CDRS-R).**

Coordinates in MNI space.Threshold p < 0.001, uncorrected, k > 10. COG, center of gravity; L, left; R, right; FA, fractional anisotropy; MD, mean diffusivity; RD, radial diffusivity; AD, axial diffusivity; WM, white matter; PHG, parahippocampal gyrus; ACC, anterior cingulate cortex; MFG, medial frontal gyrus; OFC, orbitofrontal gyrus; ILF; inferior longitudinal fasciculus; IFOF, inferior-fronto-occipital fasciculus; ATR, anterior thalamic radiation; SLF, superior longitudinal fasciculus.

the hippocampus – similar to a cluster from the group comparison analysis – and decreased in the anterior limb of the internal capsule, OFC projection fibers, and the posterior cingulum near the precuneus. Furthermore, anhedonia was positively correlated with MD in OFC projection fibers, the external capsule, and the sagittal stratum. Additionally, increased anhedonia severity was associated with greater RD in the same posterior limb of the internal capsule and posterior cingulum clusters as the FA analysis, the same OFC projection fibers as the MD analysis, and clusters in the ATR and corticospinal tract. Finally, there were positive correlations between anhedonia and AD in the corticospinal tract and projection fibers into the occipital cortex.

#### **WHOLE-BRAIN CORRELATIONS WITH IRRITABILITY IN MDD**

Analyses revealed a total of 14 uncorrected clusters, with 2 overlapping clusters (**Table 5**, **Figure 4**). As irritability increased, FA decreased in clusters in the sagittal stratum and IFOF, while MD increased in the same sagittal stratum cluster as well as in clusters in the ACR, SLF, and IFOF. For RD, positive correlations with irritability were evident in the same sagittal stratum cluster, the anterior limb of the internal capsule, and the SLF. Positive correlations were also found between AD and irritability in the same ACR cluster as the MD analysis as well as in the IFOF, the corticospinal tract, and the SLF.

### **DISCUSSION**

Consistent with our hypotheses, analyses revealed reduced WM integrity (i.e., decreased FA, and increased MD, RD, AD) in the MDD group compared to HC as well as in the more severely depressed, anhedonic, and irritable patients. Furthermore, despite significant correlations between the two dimensional measures and depression severity, we found distinct WM alterations for both anhedonia and irritability that differed from those for depression severity. Reduced integrity was found in fronto-striatal and thalamic tracts, the corpus callosum, and tracts connected to the inferior temporal (IT) cortex. Additionally, reduced integrity in the sagittal stratum was consistently found in our analyses to be correlated with increasing depression severity, anhedonia, and

irritability. Unexpectedly, analyses also revealed a cluster in the posterior cingulum near the hippocampus which demonstrated more anisotropic diffusion, both as anhedonia increased and in the MDD group vs. HC. Finally, it is interesting to note that in many of our analyses there was overlap in the clusters demonstrating a relationship with FA and RD, potentially suggesting that structural issues related to RD are driving the observed relationships with FA in this and other studies.

#### **GROUP DIFFERENCES IN WM INTEGRITY**

Group differences were observed in both the posterior and anterior cingulum. Specifically, depressed adolescents demonstrated decreased WM integrity in the anterior cingulum near the precuneus, and increased integrity in the posterior cingulum near the hippocampus, compared to HC. The cingulum connects the cingulate and entorhinal cortices and is broadly involved in attention, memory, and emotions (29, 30). The anterior portion of the cingulate has been implicated in emotional processing and depression (31). Altered functioning, connectivity, and diffusion around the precuneus are frequently reported in MDD (32, 33). Given the role of the precuneus in self-related processes, and that self-processing is typically altered in depression (34), this potentially suggests that reduced WM integrity contributes to altered functioning in this region early in the course of the disease.

The MDD group also demonstrated more coherent diffusion in the posterior cingulum near the hippocampus. The posterior cingulate is involved in cognitive functions including attention and memory (35). Functional hyperactivity in the hippocampus (36–38), as well as decreased hippocampal volume (39), are consistent findings in adult MDD. Given the role of the hippocampus in learning and memory (40), but also in the regulation of motivation and emotion (41, 42), this region is critical to carrying out normal behaviors that may be altered in depression. Furthermore, greater WM integrity in tracts leading to the hippocampus would be consistent with the literature demonstrating hyperactivity of this region in non-medicated MDD patients. Overall, the categorical comparison between depressed adolescents and HC revealed differences in an important tract connecting prefrontal and limbic regions.

### **DEPRESSION SEVERITY AND WM INTEGRITY**

Our use of an approach that accounts for a range of depression severity in our sample revealed a pattern of reduced WM integrity as depression severity increased. Specifically, we found reduced integrity in the genu of the corpus callosum, a region that connects prefrontal and orbitofrontal cortices. Many studies have documented altered diffusivity in the genu (16) as well as reduced volume (6, 43–45). Given that the prefrontal and orbitofrontal cortices are involved in critical processes, including decision-making, attention, reward processing, and the evaluation and regulation of emotion (46–48), an interruption in communication between these areas has implications for depression and mood disorders.

Additionally, we found decreased integrity in the sagittal stratum with increasing severity, not only in this analysis, but also in the dimensional analyses within the MDD population. The sagittal stratum is a complex fiber bundle connecting the occipital cortex to the rest of the brain, and includes fibers from many major tracts including the ILF and IFOF (49). The ILF and IFOF both connect the occipital cortex to temporal limbic structures (i.e., amygdala, hippocampus) and the PFC, although the IFOF connects directly

#### **Table 4 | Voxel-wise correlations with anhedonia.**

Coordinates in MNI space. Threshold p < 0.001, uncorrected, k > 10. COG, center of gravity; L, left; R, right; FA, fractional anisotropy; MD, mean diffusivity; RD, radial diffusivity; AD, axial diffusivity; WM, white matter; OFC, orbitofrontal gyrus; IC, internal capsule; ATR, anterior thalamic radiation; IFOF, inferior-fronto-occipital fasciculus; ILF, inferior longitudinal fasciculus.

to the OFC and the ILF does so indirectly through the uncinate fasciculus (50). Therefore, both tracts are involved in connecting visual information with areas involved in emotional memories, judgments, and behaviors. A meta-analysis of diffusion studies of patients with MDD found WM alterations in both the ILF and IFOF (16). Additionally, alterations in WM have been found in the IFOF for depressed adolescents (17), adolescents with a familial risk for depression (18), and adults with MDD (51).

We also observed reduced integrity with increasing severity in bilateral clusters in the ATR near the pallidum. The ATR connects thalamic nuclei with the PFC through the anterior limb of the internal capsule. Reduced WM integrity has been reported in the ATR in several studies of depressed adults (16). Furthermore, given the role of the thalamus in motivation and goal pursuit (52, 53), altered connectivity within this circuit could contribute to the motivational deficits associated with depression.

Additionally, increased illness severity was associated with reduced integrity in the corticospinal tract near the postcentral gyrus. The corticospinal tract transmits motor impulses from the motor and premotor cortices to the spinal cord. Although this was an unexpected finding, motor disturbances and retardation are a relevant clinical symptom of depression (54). In this way, altered diffusivity may be related to the observed slowing and impairment of motor functions. Finally, we again found decreased integrity with increasing severity in the previously described anterior cingulum near the precuneus, which is consistent with the findings from our group analysis. Overall, our analysis with varied levels of depression severity was more robust than the categorical comparison and revealed a more extensive network of reduced WM integrity.

#### **ANHEDONIA AND WM INTEGRITY**

The dimensional analysis with anhedonia revealed an association between increased anhedonia and reduced integrity in the anterior limb of the internal capsule near the thalamus, a tract implicated in reward processing (19). The anterior limb of the internal capsule connects the thalamus with cingulate and prefrontal cortices, which are heavily involved in motivation, decision-making, and evaluating the saliency of emotional and rewarding stimuli. Additionally,increased anhedonia was correlated with reduced integrity in tracts (i.e., IFOF, projection fibers) connected to the posterior lateral OFC (BA 47), an area involved in many functions including emotional and reward processing, complex learning, and the inhibition of responses (46, 47, 55). Depressed patients

have demonstrated reduced gray matter volume in the posterior lateral OFC as well as altered functional responses to emotional stimuli, reward processing, and reversal learning (56).

We also found reduced integrity in the external capsule as anhedonia increased. The external capsule contains cholinergic fibers projecting from the basal forebrain to the cerebral cortex. Reduced integrity in the external capsule has been found previously in adult MDD (57, 58). Furthermore, we once again found reduced integrity in the previously discussed sagittal stratum and posterior cingulum near the precuneus with greater symptom severity. Finally, the analysis revealed increased integrity with increased anhedonia in the posterior cingulum near the hippocampus, in an area fairly close to the cluster that showed increased integrity in the MDD group in our categorical comparison. In this way, it is possible that anhedonic symptoms are related to the group differences we observed. Given the previously discussed role of the hippocampus and limbic system in the regulation of motivation and emotion, the relationship between hippocampal functioning and anhedonia represents an important area for future research.

#### **IRRITABILITY AND WM INTEGRITY**

As predicted, we found decreased integrity as irritability increased in a tract near the amygdala (i.e., sagittal stratum including the ILF and IFOF). However, increased irritability was correlated with decreased integrity in tracts primarily connecting to prefrontal and occipital cortices. We also found clusters in the previously discussed IFOF, although one was in the lingual gyrus while the other was in the middle frontal gyrus. The lingual gyrus has been implicated in processing emotional faces (59), which is typically altered in MDD (60). Altered cerebral blood flow and resting state connectivity have been demonstrated in the lingual gyrus in adults with MDD (61, 62). Additionally, decreased integrity in WM has previously been found around the middle frontal gyrus (63), an area broadly involved in a variety of higher-level cognitive processes (64) which are often compromised in MDD.

Reduced integrity related to elevated irritability was also found in the ACR, which connects the striatum to the ACC. Reduced integrity has previously been demonstrated in the ACR in pediatric bipolar patients (65), and dysfunctional activity in the ACC is typically considered a hallmark of depression (37, 42, 66–70). Furthermore, altered intrinsic functional connectivity (i.e., resting state) between the striatum and ACC has been documented for depressed adolescents (8). Finally, a cluster in the SLF in the IT gyrus was found. The SLF is a major bidirectional association tract connecting large parts of the frontal cortex with the parietal, temporal, and occipital lobes. Less restricted diffusion in the SLF has been previously demonstrated for depressed adolescents (17), adolescents with a genetic risk for depression (18), and adults with MDD (71).

#### **MEASURES OF WM INTEGRITY**

Although a complete discussion of RD and AD goes beyond the scope of this paper, it is interesting to note that for both the categorical and dimensional analyses we found overlap in

#### **Table 5 | Voxel-wise correlations with irritability.**

Coordinates in MNI space.Threshold p < 0.001, uncorrected, k > 10. COG, center of gravity; L, left; R, right; FA, fractional anisotropy; MD, mean diffusivity; RD, radial diffusivity; AD, axial diffusivity; WM, white matter; IT, inferior temporal gyrus; MFG, medial frontal gyrus; MT, middle temporal gyrus; ILF, inferior longitudinal fasciculus; IFOF, inferior-fronto-occipital fasciculus; ACR, anterior corona radiata; ATR, anterior thalamic radiation; SLF, superior longitudinal fasciculus; IC, internal capsule.

**FIGURE 4 | DecreasedWM integrity as irritability increased in the (A) sagittal stratum in the IT; (B) IFOF near the MFG**. Key: IT, inferior temporal cortex; IFOF, inferior-fronto-occipital fasciculus; MFG, medial frontal gyrus.

clusters with reduced FA and increased RD, but no overlapping relationships with AD. Increased RD may be caused by disturbances in myelin, whereas decreased AD has been suggested to reflect disrupted axonal integrity (72–74). As such, our findings and those from previous research may reflect that alterations in FA for MDD are being driven more by issues of myelination than axonal integrity. However, further research is needed to replicate and expand upon a possible mechanism.

#### **LIMITATIONS AND FUTURE DIRECTIONS**

Although our findings are consistent with other clinical studies investigating altered WM in depressed adolescents, it should be noted that very liberal thresholds were used for the analyses and the inability to correct for multiple comparisons is an issue of concern. Although our statistical methodology and sample size were comparable to those of other studies of clinical populations using DTI (17, 63, 75), it is possible that the sample sizes used in many clinical studies are not large enough to produce adequate statistical power. In this way, it is difficult to adequately balance the concerns of committing a Type I error by not correcting while also avoiding a Type II error due to small sample sizes and reduced statistical power. Therefore, our findings are considered preliminary. Furthermore, the inclusion of patients with milder symptomatology may have weakened our ability to detect group differences.

Although small sample sizes may be a possible explanation for the relatively weak results in our and other clinical studies of adolescent depression, another possibility is that the adolescent brain is still malleable and the alterations in WM structure may not fully take hold until adulthood (11). Therefore, it is even more pressing to understand a neuroimmunological model of depression and the factors that may contribute to changes in WM before chronicity begins to take effect. For example, given past findings that depressed adolescents exhibit higher levels of circulating inflammatory cytokines (76), one possible explanation for the observed reduction in FA in adult MDD is that it may reflect effects of chronic low grade inflammation. Additionally, given our previous research on fronto-striatal functional connectivity in MDD,future studies should investigate altered WM microstructure using a targeted tractography approach. Finally, further research is needed to investigate this hypothesis and other models of the systemic consequences of depression. To this end, a better understanding of what FA, MD, AD, and RD illustrate in an adolescent population, as well as the factors that contribute to these diffusivity measures, is needed in the field.

#### **CONCLUSION**

Our investigation of altered WM microstructure in medicationfree adolescents with MDD revealed a general pattern of impaired WM integrity in the depressed adolescents, and as depression severity, anhedonia, and irritability increased. Our findings are consistent with an overall hypothesis that depression, even in adolescence, involves a disconnection of prefrontal, striatal, and limbic emotional areas (16). Although this represents a good step toward understanding depression during this critical period, more research is needed to understand the factors that ultimately contribute to alteredWM microstructure in order to develop potential interventions.

#### **ACKNOWLEDGMENTS**

We would like to thank Chao-Gan Yan of the Child Mind Institute for his help with this project. This study was supported by grants from the NIH (AT004576, MH095807), the Brain & Behavior Research Foundation (formerly NARSAD), the Hope for Depression Research Foundation, and generous gifts from the Leon Levy Foundation.

### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 04 October 2013; accepted: 08 November 2013; published online: 25 November 2013.*

*Citation: Henderson SE, Johnson AR, Vallejo AI, Katz L, Wong E and Gabbay V (2013) A preliminary study of white matter in adolescent depression: relationships with illness severity, anhedonia, and irritability. Front. Psychiatry 4:152. doi: 10.3389/fpsyt.2013.00152*

*This article was submitted to Neuropsychiatric Imaging and Stimulation, a section of the journal Frontiers in Psychiatry.*

*Copyright © 2013 Henderson, Johnson, Vallejo, Katz, Wong and Gabbay. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Meta-analyses of developing brain function in high-risk and emerged bipolar disorder

#### **Moon-Soo Lee1,2, Purnima Anumagalla1 , Prasanth Talluri 1 and Mani N. Pavuluri 1\***

1 Pediatric Brain Research and Intervention Center, University of Illinois at Chicago, Chicago, IL, USA

2 College of Medicine, Korea University, Seoul, South Korea

#### **Edited by:**

Stephanie Ameis, The Hospital for Sick Children and University of Toronto, Canada

#### **Reviewed by:**

Meng-Chuan Lai, University of Cambridge, UK Nicholas Neufeld, University of Toronto, Canada Annette Beatrix Bruehl, University of Cambridge, UK

#### **\*Correspondence:**

Mani N. Pavuluri, Pediatric Brain Research and Intervention Center, University of Illinois at Chicago, M/C 747, 1747 West Roosevelt Road, Chicago, IL 60608, USA e-mail<EMAIL_ADDRESS>
**Objectives:** Identifying early markers of brain function among those at high risk (HR) for pediatric bipolar disorder (PBD) could serve as a screening measure when children and adolescents present with subsyndromal clinical symptoms prior to the conversion to bipolar disorder. Studies on the offspring of patients with bipolar disorder who are genetically at HR have each been limited in establishing a biomarker, while an analytic review in summarizing the findings offers an improvised opportunity toward that goal.

**Methods:** An activation likelihood estimation (ALE) meta-analysis of mixed cognitive and emotional activities using the GingerALE software from the BrainMap Project was completed. The meta-analysis of all fMRI studies contained a total of 29 reports and included PBD, HR, and typically developing (TD) groups.

**Results:**The HR group showed significantly greater activation relative to theTD group in the right DLPFC–insular–parietal–cerebellar regions. Similarly, the HR group exhibited greater activity in the right DLPFC and insula as well as the left cerebellum compared to patients with PBD. Patients with PBD, relative to TD, showed greater activation in regions of the right amygdala, parahippocampal gyrus, medial PFC, left ventral striatum, and cerebellum and lower activation in the right VLPFC and the DLPFC.

**Conclusion:** The HR population showed increased activity, presumably indicating greater compensatory deployment, in relation to both the TD and the PBD, in the key cognition and emotion-processing regions, such as the DLPFC, insula, and parietal cortex. In contrast, patients with PBD, relative to HR and TD, showed decreased activity, which could indicate a decreased effort in multiple PFC regions in addition to widespread subcortical abnormalities, which are suggestive of a more entrenched disease process.

**Keywords: pediatric bipolar disorder, high risk, meta-analysis, GingerALE, dorsolateral prefrontal cortex, amygdala**

### **INTRODUCTION**

The relationship between pediatric and adult bipolar disorder has been the subject of controversy. It is not clear whether pediatric bipolar disorder (PBD) is the pediatric form of the typical adult bipolar disorder or an entity of its own, as bipolar disorder usually manifests differently in childhood than in adulthood. Some studies in adults have reported that a portion of adults with bipolar I disorder experienced childhood or adolescent onset, and some of them began showing symptoms even before 12 years of age (1, 2). Identifying early markers of brain function among those at high risk (HR) for PBD could serve as a screening measure when children and adolescents present with subsyndromal clinical symptoms prior to the conversion to bipolar disorder (pediatric or adult form). These biomarkers can also aid as a stand-alone bio-signature for the identification of risk even prior to the emergence of any clinical symptoms and could allow an opportunity to prevent the onset of full-blown illness (3). One way to begin identifying the biomarkers is to examine the brain function in the genetically HR offspring of patients with bipolar disorder. While some studies of HR have been published (4–11), due to

their small sample sizes and corrections for multiple comparisons, the findings remain inconclusive.

To offer robust and reliable findings, we used a recently developed activation likelihood estimation (ALE) technique. This method assumes that the peak co-ordinates reported by each study represent the activation maps from which they are derived and uses the reported co-ordinates in voxel-wise analysis to assess the consistency of activation in any given set of studies (12–14). By performing the quantitative voxel-wise meta-analysis of already published results from the HR population and comparing them with those from the converted PBD and typically developing (TD) youth, we can provide objective, unbiased, and statistically based quantified evidence.

Ideally, a separate meta-analysis would be conducted for each individual domain, such as emotion processing or attention, as they relate to bipolar disorder diathesis. However, given the infancy of the current literature regarding HR patients, this is not practical, as no individual construct has included a sufficient number of studies to date. Instead, it is more feasible to study the commonalities probed across multiple domains in a systematic and

statistically driven fashion. There is a certain advantage to combining all the studies that include multi-domain probes. First, the brain does not work in isolation across individual domains; therefore, it is necessary to examine the brain's function as a whole while it is engaged in affective, cognitive, and motor control tasks (15). Furthermore, pooling several pilot studies produces an exploratory power of how the brain functions in a larger sample, eventually offering the possibility of correlating the results with the clinical manifestations of domains and disorders presenting with combined affective, cognitive, and motoric symptoms (16). This approach is a segue into future studies that can explore the interface of multiple domain functions in individual studies.

We consider emotional systems and circuits, in illness or wellness, to be closely linked to cognitive and motor control circuits of attention, working memory, and response inhibition (17). These systems interface at three tiers as shown in animal (18) and human studies of PBD (19): (1) at the prefrontal level between the ventrolateral prefrontal cortex [VLPFC; inferior frontal gyrus; Brodmann areas (BAs) 45, 47] and the dorsolateral prefrontal cortex (DLPFC; middle frontal gyrus; BAs 9, 9, 46), (2) at the intermediary cortex in the anterior cingulate cortex (ACC), such as between the dorsal (BA 32) and pregenual ACC (BA 24), and (3) at the subcortical level between the amygdala and striatum (19). While we could not determine which probe or domain dysfunction would contribute to activity in any given co-ordinate in this meta-analysis, we developed our hypotheses based on knowledge derived from the emerging literature. Emotion-processing tasks probing the affective systems entered into our meta-analysis would contribute to the increased prefrontal activity at the interface of VLPFC and DLPFC in HR and the decreased activity in PBD relative to TD (19). Increased subcortical amygdala activity would be a specific marker of PBD (20) relative to HR and TD. Based on our knowledge of attention and working memory task response, the DLPFC will manifest with increased activity in HR (6) and decreased activity in PBD (21),relative to TD. Impaired subcortical striatal activity would be a more entrenched specific marker of PBD's cognitive and motor dysfunction (20, 22, 23) relative to the HR and TD groups.

### **MATERIALS AND METHODS SEARCH STRATEGY**

We identified primary studies through a comprehensive literature search of the MEDLINE (using both free-text and MeSH search) and PsychINFO databases using the following keywords: pediatric or child or adolescent, plus bipolar disorder or highrisk or at risk, and plus functional magnetic resonance imaging or fMRI. In addition, manual searches were conducted via reference sections of review articles and individual studies to check for any missing studies that were not identified using computerized searches. There were no language restrictions; in fact, all the included manuscripts were written in English. Only fMRI studies were chosen for review. An initial list of studies was produced that included any report of fMRI studies of PBD and HR offspring published in print or online by December 31, 2013. The selection process for the final list of primary studies for the planned meta-analyses in this study was very specific. The first-level literature search yielded 235 unique published articles with 49 studies meeting the initial inclusion criteria. A further manual search leads to eight other studies. After a second-level review of these 57 studies, only 29 contained the co-ordinates essential for inclusion in our meta-analysis (**Figure 1**). Any ambiguity in inclusion was resolved through a consensus decision by the authors of this manuscript. Study data (e.g., co-ordinates, participant numbers, and imaging spaces) were entered and crosschecked by participating authors.

### **SELECTION CRITERIA**

"High risk" in this project refers to adolescents who have a biological parent diagnosed with BD. We selected studies with participants whose mean age was less than 19 years. Every study that we included had participants between the ages of 7 and 18 except for the study performed by Thermenos et al. (11), where the ages ranged up to 24. All reports included in the meta-analysis satisfied the following criteria: (1) a healthy comparison group is included, (2) the studies conducted whole-brain analyses, (3) all studies provided standard Talairach or Montreal Neurological Institute (MNI) spatial co-ordinates for the key findings, (4) patient participants had been diagnosed with bipolar disorder, and (5) there were at least five members in each of the participant groups. We included only those studies that reported activation foci as 3D co-ordinates in stereotactic space, examined active task constructs, and presented results for groups of participants.

Excluded manuscripts consisted of the following: (1) reviews or meta-analyses, (2) those with subject overlap, and (3) other MRI modalities (e.g., structural imaging, spectroscopy, diffusion tensor imaging, and functional connectivity studies).

#### **ACTIVATION LIKELIHOOD ESTIMATION METHODS AND PAIRWISE ALE META-ANALYSIS**

GingerALE software version (version 2.3.1) from the BrainMap project was used to conduct ALE meta-analysis of eligible studies (13, 14, 43). Meta-analyses were performed using the revised ALE software (i.e., GingerALE 2.3). The key modification in the revised ALE software included the change from fixed-effects (convergence between foci) to random-effects inference (convergence between studies but not individual foci reported for the same study), as well as greater meta-analytic weighting for primary studies that involved more participants. In line with our goal of gaining insight on the whole brain's function through tasks that probe combined domains, we performed exploratory analyses using all eligible data in the HR offspring, BD patient, and TD groups in the pediatric age group. Conversely, we did not separate the analyses by the type of the task or the brain domain probed. This method also helped to harness sample size and power. Activation co-ordinates reported in the MNI space were converted to Talairach co-ordinates using the Lancaster transform (icbm2tal) in GingerALE. Our meta-analysis was conducted in Talairach space. Co-ordinates originally presented as MNI space were transformed into Talairach space using Lancaster transformation. For uniformity, Talairach co-ordinates expressed by the previous Brett transformation (44) were converted into MNI space and re-transformed into Talairach space. The meta-analysis was performed using pairwise ALE meta-analysis.

Pairwise ALE meta-analyses included the following comparisons at first: greater activation in PBD versus HR, in HR versus PBD, in PBD versus TD, in TD versus PBD, in HR versus TD, and in TD versus HR. However, two pairwise ALE meta-analyses (greater activation in PBD versus HR and greater activation in TD versus HR) were not performed due to the lack of available data. The input co-ordinates were weighted to form estimates of activation likelihood for each intracerebral voxel. The activation likelihood of each voxel in standard space was then combined to form a statistic map of the ALE score at each voxel. Statistical significance of the ALE scores was determined by a permutation test controlling the false discovery rate (FDR) at *p* < 0.05 (45). The statistic maps were thresholded by default at this critical value, and a recommended minimum cluster size was suggested from the cluster statistics. By using this minimum cluster size for the supra-threshold voxels, we can obtain the thresholded ALE image. Pairwise ALE analyses results were reported at *p* = 0.05 and were whole-brain corrected. A Talairach Daemon was used for anatomical locations for significant clusters.

### **RESULTS**

The meta-analysis of all fMRI reports included 29 studies (PBD, HR, and TD). There was no overlap in patients who completed the same task across the selected studies. The primary studies included in the meta-analysis are listed in **Table 1**. Findings are summarized in **Table 2** and **Figure 2**.

#### **HR AND TD: RECOGNIZING HIGH-RISK PARTICIPANTS**

Participants in the HR group showed significantly greater activation in the right DLPFC, insula, inferior parietal lobule, and left cerebellum relative to TD. No other group differences were found. In case of greater activation in the TD group relative to HR, the analysis was not performed due to the lack of a large enough sample size and of experiments showing significant results.

### **PBD AND HR: RECOGNIZING THE EMERGENCE OF THE DISORDER**

The HR group showed significant greater activation of the right DLPFC, insula, and left cerebellum than PBD. No other group differences were identified. In case of greater activation in the PBD group relative to HR, the analysis was also not performed due to a small sample size and few experiments showing significant results.

#### **PBD AND TD: RECOGNIZING THE ILLNESS FROM WELLNESS**

Patients with PBD demonstrated greater activation in the subcortical regions of the right amygdala, the parahippocampal gyrus, the subgenual ACC, and the medial PFC, and in the left ventral striatum, VLPFC, and cerebellum relative to TD. The TD group showed greater activation in the right VLPFC, DLPFC, superior frontal gyrus, dorsal ACC, and striatum than patients with PBD.

### **DISCUSSION**

We found the recently published developmental meta-analysis of bipolar disorder performed by Wegbreit et al. The researchers compared different age groups with bipolar disorder (youths and adults). PBD youths showed increased activation in the amygdala, the inferior frontal gyrus, and precuneus compared to bipolar disorder adults during tasks using emotional stimuli. These findings revealed that these structures are underdeveloped and work less efficiently when compared with those of adults (46). However, our meta-analysis was conducted using the comparison between participants of the same age (participants' mean age is less than 19 years). The central findings of the meta-analyses of brain function among the PBD, HR, and TD groups, during the performance of mixed cognitive and emotional activities, illustrated a coherent pattern of group differences in line with our *a priori* hypothesis. The HR group showed a significantly greater activation in the *right DLPFC–insular–parietal–cerebellar regions* relative to TD, and this may be a bio-signature – an earlier sign of potential PBD development. At the junction of the DLPFC and VLPFC regions, where prefrontal systems interface in voluntary modulation of cognition, emotion, and motor control, brain function was amplified in the HR group (6, 7). Large future studies of symptomatic HR population (47) and genetic HR population must be compared both at a symptomatic and brain functional level to look at the definitive predictability of symptoms and the correlation of brain activity patterns.

#### **Table 1 | Primary fMRI studies of participants with pediatric bipolar disorder (PBD), those at high risk (HR) for PBD, and typically developing (TD): children included in meta-analysis**.

(Continued)

#### **Table 1 | Continued**

aStudies including HR groups.

Some studies were missing age range information and showed only the mean age. Accordingly, that information could not be included within the table. Specific medications were heterogeneous when reported and at times went unreported. Hence, we were only able to comment on participants' medicated/unmedicated status. Similarly, the mood and affect of participants were also largely unreported and, therefore, could not be included in the table.

A repeated and important observation of hemodynamics of the fMRI studies is the increased activity in the brain that reflects increased effort (48). If one construes TD as the reference point of normative activity, then the HR group showed increased effort to get the same work done by deploying the right DLPFC–insular– parietal regions relative to TD, while in PBD, these same regions went offline relative to TD. This finding is akin to the analogy of "stretching an elastic band"with increased DLPFC activity (requiring a greater effort than TD) in the HR group, whereas those with PBD who had a more severe illness had reached a breaking point with decreased right VLPFC and DLPFC activity (with no effort to spare relative to TD). We could not explain the increased left VLPFC activity in PBD relative to TD. While such a finding is not unexpected in a meta-analytic study, it was largely based upon the

**Table 2 | Activation likelihood estimation (ALE) meta-analysis findings for fMRI studies comparing pediatric bipolar disorder (PBD) patients, participants with a high risk (HR) for PBD, and typically developing (TD) children**.

R: right, L: left.

participants of only one study (21). However, it can be explained by bilateral disturbances in the VLPFC in PBD, albeit with the common and prominent right-sided abnormality than the left (32, 37). In the end, while one can postulate with explanations consistent with repeatedly published findings, definitive interpretations are not possible in understanding the nature of abnormal hemodynamic activity. For example, decreased (5) or increased (6) activation of the striatum with failed trials cannot easily differentiate HR from PBD based on any individual study. It could be mediated by the severity of illness in case of PBD, subsyndromal symptoms in HR, type of task, or hemodynamic relationship between the striatum and the PFC control regions.

With regard to recognizing the fully formed illness, typically noted underactivity of the higher cortical regions of emotion modulation (i.e., the interfacing dyad of the right VLPFC and DLPFC in the prefrontal regions) and overactivity of the subcortical amygdala consistently reported in BD Type I participants relative to TD adolescents (19) has also emerged as a significant finding in the current meta-analyses. The VLPFC is believed to serve the dual function of emotion (49) and motor (50) control via top–down regulation of the amygdala (51) and striatum (52), respectively. The DLPFC also serves a dual function, but it is predominantly through diverse cognitive functions involving executive control, response selection, problem solving, and emotion (53), and by being closely connected to the medial PFC, VLPFC, and the subcortical regions directly (54) as well as indirectly (52). The cognitive and emotion control regions in the PFC are not able to moderate the overactive subcortical regions, a consistent finding that was further underscored in our meta-analysis. In addition to the top–down *affect modulation circuitry* problems, increased activity is lateralized to the left side in the evaluative medial PFC, pregenual ACC, and the striatal loop (55); furthermore, all these regions are known to be closely connected to the amygdala (56). This subcortical and medial PFC loop is the *affective evaluation circuit* that is overactive in PBD. These findings could explain the excessive reactivity to negative emotions reported in PBD (21, 57) and are also in line with the concept suggested for bipolar disorder in general, including adult patients. Phillips and Swartz conceptualized bipolar disorder as multiple dysfunctions in prefrontal hippocampal–amygdala, emotion processing,

and emotion-regulation circuits, together with an "overactive," left-sided ventral striatal-ventrolateral, and orbitofrontal cortical reward-processing circuit (58). These results attest to the fact that, in relative terms of group comparison from fMRI studies, cognitive

DLPFC and the corresponding dorsal circuitry hub that includes the parietal region and the insula are more involved in the HR population, while the wider multiple cortico (VLPFC, DLPFC, and

**(A)** DLPFC: dorsolateral prefrontal cortex, x = 46, y = 8, z = 22, cluster

medial PFC) and subcortical (limbic and basal ganglia) regions are

ventrolateral prefrontal cortex.

implicated in PBD. Published structural and fMRI studies of HR have not been conclusive and are limited to a comparison with the TD at times (7, 11). Singh et al. (59) reported that 8- to 12-year-old children with a familial risk for mania did not exhibit any statistically significant volumetric differences in the PFC, thalamus, striatum, or amygdala

compared with the TD group. However, they concluded that longitudinal studies will be needed to examine whether structural changes over time may be associated with a HR for BD (59). Bechdolf et al. (60) reported volume reduction in emotion-processing regions (i.e., the insula and amygdala) in HR, relative to TD, that corresponded to the functional abnormality involving increased amygdala activity in HR (9). While we found abnormal function in the insula in HR in this meta-analysis, three-way comparison did not reveal increased amygdala activity in HR. Existing studies consistently reported smaller amygdala and hippocampus (61), larger basal ganglia (62), and reduced PFC gray matter (63) in PBD. Hemodynamic (64) and resting state connectivity (65) findings in PBD relative to TD also point to frontolimbic and frontostriatal functional disturbance in PBD. Such uniformity in multi-modal imaging findings attests to the high reliability in establishing a significant pattern of brain dysfunction specific to PBD.

Limitations of this study include fewer and unequal numbers of participants in the HR group and the inclusion of studies that employed variable tasks used to probe multiple domains. However, due to the broad array of daily functions that draws from the active involvement of multiple and highly integrated networks, and the dual engagement of VLPFC, DLPFC, ACC, and the striatum in both cognitive and emotional tasks, this study was a reasonable first attempt to examine the entire brain's level of functionality from the existing data.

#### **ACKNOWLEDGMENTS**

We thank all the authors of the included published papers for their important contributions to the field. Support for this work was provided by endowed funds by the Colbeth Foundation and Berger-Colbeth Chair funds. All authors had full access to the data in the study. Dr. Pavuluri takes responsibility for directing the analysis and interpretation of the data. Dr. Moon-Soo Lee ensured the accuracy of the data analysis. Drs. Anumagalla, Talluri, and Lee entered the data and checked them for accuracy. Inclusion and exclusion criteria were applied following the full consensus of all authors.

#### **REFERENCES**

64. Wegbreit E, Passarotti AM, Ellis JA, Wu M, Witowski N, Fitzgerald JM, et al. Where, when, how high, and how long? The hemodynamics of emotional response in psychotropic-naive patients with adolescent bipolar disorder.*J Affect Disord* (2013) **147**(1–3):304–11. doi:10.1016/j.jad.2012.11.025

65. Wu M, Lu LH, Passarotti AM, Wegbreit E, Fitzgerald J, Pavuluri MN. Altered affective, executive and sensorimotor resting state networks in patients with pediatric mania. *J Psychiatry Neurosci* (2013) **38**(4):232–40. doi:10.1503/jpn. 120073

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 10 June 2014; accepted: 24 September 2014; published online: 03 November 2014.*

*Citation: Lee M-S, Anumagalla P, Talluri P and Pavuluri MN (2014) Meta-analyses of developing brain function in high-risk and emerged bipolar disorder. Front. Psychiatry 5:141. doi: 10.3389/fpsyt.2014.00141*

*This article was submitted to Neuropsychiatric Imaging and Stimulation, a section of the journal Frontiers in Psychiatry.*

*Copyright © 2014 Lee, Anumagalla, Talluri and Pavuluri. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Stress, inflammation, and cellular vulnerability during early stages of affective disorders: biomarker strategies and opportunities for prevention and intervention

#### **Adam J.Walker 1,2,Yesul Kim1,2, J. Blair Price1 , Rajas P. Kale1,3, Jane A. McGillivray 2 , Michael Berk 4,5,6,7 and Susannah J. Tye1,2,8\***

1 Department of Psychiatry and Psychology, Mayo Clinic, Rochester, MN, USA

2 School of Psychology, Deakin University, Melbourne, VIC, Australia

3 School of Engineering, Deakin University, Geelong, VIC, Australia

4 School of Medicine, Deakin University, Geelong, VIC, Australia

5 Department of Psychiatry, University of Melbourne, Melbourne, VIC, Australia

6 Orygen Youth Health Research Centre, Melbourne, VIC, Australia

7 The Florey Institute of Neuroscience and Mental Health, Melbourne, VIC, Australia

8 Department of Psychiatry, University of Minnesota, Minneapolis, MN, USA

#### **Edited by:**

Stephanie Ameis, University of Toronto, Canada

#### **Reviewed by:**

Vilma Gabbay, Mount Sinai School of Medicine, USA Ellen Grishman, University of Texas Southwestern Medical Center, USA

#### **\*Correspondence:**

Susannah J. Tye, Department of Psychiatry and Psychology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA e-mail<EMAIL_ADDRESS>
The mood disorder prodrome is conceptualized as a symptomatic, but not yet clinically diagnosable stage of an affective disorder. Although a growing area, more focused research is needed in the pediatric population to better characterize psychopathological symptoms and biological markers that can reliably identify this very early stage in the evolution of mood disorder pathology. Such information will facilitate early prevention and intervention, which has the potential to affect a person's disease course.This review focuses on the prodromal characteristics, risk factors, and neurobiological mechanisms of mood disorders. In particular, we consider the influence of early-life stress, inflammation, and allostatic load in mediating neural mechanisms of neuroprogression. These inherently modifiable factors have known neuroadaptive and neurodegenerative implications, and consequently may provide useful biomarker targets. Identification of these factors early in the course of the disease will accordingly allow for the introduction of early interventions which augment an individual's capacity for psychological resilience through maintenance of synaptic integrity and cellular resilience. A targeted and complementary approach to boosting both psychological and physiological resilience simultaneously during the prodromal stage of mood disorder pathology has the greatest promise for optimizing the neurodevelopmental potential of those individuals at risk of disabling mood disorders.

**Keywords: prodrome, depression, bipolar, biomarker, stress, inflammation, cellular resilience, plasticity**

#### **INTRODUCTION**

There is increasing appreciation for the need to both identify and treat mood disorders during their earliest stages (1). Although some dispute remains, maladaptive changes in mood and behavior first become evident during the prodromal period (2). However, the low specificity of these changes makes the prodromal stage difficult to definitively characterize prior to disease onset (3). Observable changes in mood and general physiologic functioning can include increases in sadness, anhedonia, irritability, anger, and anxiety, together with alterations in sleep and energy (4). Correlating these symptoms with prodromal biomarkers offers an exciting juncture whereby targeted interventions could be opportunistically employed to prevent neurodegenerative changes from accruing as the disease progresses (5). The potential to intervene during the prodromal stage of psychiatric illness through the detection and remediation of novel biomarkers has perhaps been best studied in schizophrenia, wherein most individuals experience a lengthy prodromal period prior to the full emergence of diagnosable psychotic symptoms (6). As an exemplar, low levels of nervonic acid appear to be a risk factor for conversion from

high-risk to frank psychosis (7), and this risk of conversion may be reduced by targeted omega-3 fatty acid supplementation (8). Encouraging results from this work have renewed interest in the early detection of affective disorders, particularly bipolar disorder, with the hope that earlier and more targeted interventions might slow disease progression (3, 9–12). This can significantly impact neuroprogression and subsequent disease course for the individual (13). This concept of "neuroprogression" refers to the cumulative restructuring of the central nervous system which in turn mediates the development and persistence of psychiatric illness (14, 15). This process results from disturbances in inflammatory mediators, neurotrophins, oxidative stress, and energy regulation (14, 15).

#### **BIOMARKER STRATEGIES FOR PRODROMAL MOOD DISORDERS**

#### **STRESS AND ALLOSTATIC LOAD Stress sensitization and early detection**

Stress is one of the best-studied mediators by which genetic vulnerabilities are translated into mood disorder pathology through the process of neuroprogression (16–18). Numerous studies have demonstrated that both depression and bipolar disorder are more prevalent in individuals who have experienced adverse early-life events. This is partly because such experiences prime future physiologic and neural responses to stress, elicit a state of chronic inflammation (19), alter cellular mediators of plasticity and energy metabolism, and increase cellular "wear and tear" (20–22). Earlylife stress (2) can be particularly deleterious because of its potential to influence the programing of the hypothalamic–pituitary– adrenal (HPA) axis (23) to induce persistent sensitization of neuroendocrine, autonomic, oxidative, and immune responses to stress. Over time these sensitized systems cumulatively contribute to the cellular and synaptic alterations underlying neuroprogression (21, 24–26). Specific examples include changes in reactivity of inflammatory cytokines [e.g., interleukin 6 (IL-6)] (25), alterations in markers for lipid peroxidation [e.g., 8-iso-prostaglandin F (2α)],oxidative damage to DNA (8-hydroxy-20 -deoxyguanosine) and RNA (8-hydroxyguanosine) (24), as well as altered cortisol, adrenocorticotropic hormone, and corticotrophin releasing factor responses (26). Identification of the state of physiologic and cellular resilience or sensitivity to stress may provide an important indicator of the level of neuroprogression and stress-mediated disease pathology for affective disorders,potentially prior to the initial manifestation of the mood episode (22).

One mechanism whereby HPA axis sensitization is likely to occur is through epigenetic regulation of stress response processes (21, 27). Evidence shows that exposure to various forms of stress result in multiple epigenetic changes in limbic regions as well as the HPA axis (21, 27). Interestingly, a recent study by Klendel and colleagues (18) found that only individuals who exhibited allele-specific DNA demethylation in functional glucocorticoid response elements of FK506 binding protein 5 (*FKBP5*), were prone to developing persistent cortisol dysregulation (18, 21). Further, this association was found to be dependent on an interaction effect with trauma in early life, suggesting that key developmental stages are directly related to stability of the observed effects across time (18). In another study, significant interactions between peripheral *FKBP5* mRNA expression and disease progression were reported, suggesting that polymorphisms in the gene directly impact the extent of neuroendocrine dysregulation, and corresponding neuroprogression (28). The *FKBP5* risk allele and corresponding levels of mRNA expression may represent useful biomarkers. These markers could be employed to identify individuals in the prodromal stages of stress-sensitive psychiatric disorders, such as major depression or bipolar disorder. Such detection would facilitate early intervention and could improve resilience and alleviate allostatic load in the prodromal individual.

#### **Early-life stress and accumulation of allostatic load**

Accumulation of allostatic load is a key mechanism through which early-life stress is thought to result in psychopathology (29). This is mediated via a series of enduring adaptive changes across a range of systems primed both to respond rapidly to challenge, as well as to restore homeostatic equilibrium (30). Adaptive allostatic mechanisms may fail when chronically challenged or when regulatory systems falter. This leads to a state of allostatic overload, which is thought to considerably impact the clinical course of mood disorders (31–33). Without sufficient opportunity for recovery, the brain and body are repeatedly exposed to molecular mediators of stress that can increase the level of cellular "wear and tear" (33). These mediators, which include metabolic factors, inflammatory cytokines, neurotrophins, and oxidative species, collectively impact an individual's mental and physical resilience as outlined below [for more detailed reviews see Ref. (6, 34, 35)]. Both physiological (i.e., immune and/or metabolic) and psychological (i.e., bullying) stressors contribute significantly to allostatic load, and thus need to be considered together when assessing both risk and relative staging of mood disorder pathology (6, 34).

Enhancing an individual's capacity to buffer the physiologic toll that accumulates through allostatic overload should be considered an important early intervention strategy. As allostatic load accumulates and attempts to maintain cellular homeostasis fail, cell danger signals are propagated and pro-apoptotic cell signaling pathways become increasingly engaged (36–39). This may play a role in medical comorbidities such as heart disease (40), as well as interfere with the therapeutic mechanisms of antidepressants and mood stabilizers to impair treatment efficacy (41–43). Internal stressors that activate the HPA axis and associated allostatic systems can limit an individual's capacity for allostasis even prior to the onset of external stressors (36). For example, an endogenous load can build through the expression of homocysteine or inflammatory cytokines,limiting the capacity of adaptive responses in the face of subsequent stressors. Interventions that counter this load and reduce levels of proinflammatory mediators or interfere with their neuromodulatory actions could limit neuroprogression in both bipolar and unipolar depression, as well as enhance capacity for antidepressant efficacy (44–46).

#### **INFLAMMATORY PROFILE**

Stress during earlier life is not only associated with disruption of the HPA axis, but may also serve to sensitize proinflammatory responses to future insults (47–49). Inflammatory mechanisms are increasingly appreciated for their critical role in mood disorder pathophysiology, in particular via their regulation of neuronal excitability, synaptic transmission, synaptic plasticity and neuronal survival (41, 50, 51). Of specific interest are proinflammatory mediators, such as cytokines [i.e., interleukin 1, IL-6, and tumor necrosis factor alpha (TNF-α)] and C-reactive protein (CRP). CRP is often used as a biomarker for inflammation in studies due to its relationship with proinflammatory cytokines and role in the immune response. As demonstrated by Slopen and colleagues (49), individuals at ages 10 and 15 who reported adverse life events at critical stages between the ages of 1.5 and 8 years were found to have significantly increased levels of CRP and IL-6. These heightened concentrations were correlated with immune activation and depressive-like symptoms. Notably, increased CRP levels have been used previously to predict depression severity and recurrence rates in males (48, 52).

There is a growing literature supporting the use of inflammatory biomarkers as predictors of ensuing mood disorder pathology (22). Research to date has been focused on investigating the relationship between inflammatory cytokines and affective disorders in adults; however, their specific role in early onset/adolescent psychopathology is less well explored (53). Cytokines are thought to influence neurodevelopment during key stages, such as adolescence, interacting with biological systems including those of stress hormones and gonadal hormones (53). As such, perturbation of inflammatory balance in adolescents may significantly contribute to neuroprogression and development of psychiatric illness (19, 53, 54). For example, elevated serum levels of TNF-α, IL-6, and interleukin-10 (IL-10) have been reported during the early stages of bipolar disorder (55), and CRP appears to be a biomarker of *de novo* depression risk (56).

As the mood disorder pathology progresses, an increasing number of proinflammatory cytokines are observed, including elevated levels of interferon gamma (IFN-γ) (22, 54, 55). Notably, increases in IFN-γ are associated with dysregulation of the tryptophan metabolite pathway via direct role in indoleamine 2,3-dioxygenase (IDO) activation. Activation of IDO is commonly found in later stages of mood disorders, and is a biomarker of depression-like behavior mediated by neural inflammation in animal models (48). Proinflammatory cytokines activate IDO, resulting in depletion of serotonin and augmentation of quinolinic acid (QUIN) metabolism over kynurenic acid (KYNA). Tryptophan metabolites (kynurenine, KYNA, 3-hydroxykynurenine, and QUIN) act as neuromodulators to influence behavioral, neuroendocrine, and neurochemical aspects of depression (57–60). Consequently, this accumulation of QUIN facilitates neurodegeneration over neuroprotection, impacting mood disorder neuroprogression and resultant disability (61).

It is noteworthy to mention several other findings regarding altered inflammation in youth with psychiatric pathology. Increased mRNA and protein expression levels of IL-1β, IL-6, and TNF-α were reported in the anterior prefrontal cortex of adolescent suicide victims compared with normal control subjects (62). Elevated levels of inflammatory cytokines (among others: TNF-α, IL-1β, IL-6, and IFN-γ) were also observed in the serum of pediatric patients who experienced first-episode psychosis, in addition to increased leukocyte counts and evidence of blood–brain barrier damage (63). Quantification of inflammatory biomarkers (e.g., TNF-α, IL-6, IL-10, or CRP) may thus prove useful for detecting individuals at risk for developing a mood disorder. A recent study by Byrne and colleagues (64) suggests that levels of peripheral cytokines (e.g., IFN-γ) and CRP in salivary samples may correlate with serum samples in young people. Salivary assay may prove to be a simpler, less invasive method of estimating peripheral levels of inflammatory markers in adolescents (64). This provides one avenue whereby prodromal individuals could potentially be identified and their disease onset delayed.

#### **DIMINISHED SYNAPTIC INTEGRITY**

Homeostatic control of synaptic connections within key moodrelated circuits plays a critical role in the etiology of mood disorders (65). Stress and inflammation as discussed in previous sections are implicated in disruption of synaptic signaling and integrity during the early stages of mood disorder pathogenesis. This is mediated in part through the inhibition of neurotrophin function, of which brain derived neurotrophic factor (BDNF) is the most thoroughly characterized. BDNF plays an important role in neuronal development, survival, and function, including activity-dependent synaptic plasticity (66). Synaptic plasticity is characterized by various processes, including synaptic remodeling, synaptogenesis, long-term potentiation, and long-term depression, all of which critically mediate the flow of electrochemical information throughout the central nervous system (67, 68). Stress, allostatic load, inflammation, antidepressants, and mood stabilizers exert major effects on signaling pathways that regulate cellular plasticity, suggesting these are critical neurobiological mediators of mood dysfunction and therapeutic intervention (69–72).

Glycogen synthase kinase-3 (GSK-3), part of the signaling cascade regulated by BDNF, plays an important role in synaptic homeostasis through regulation of synaptic deconsolidation (pruning) and glutamate receptor cycling (73). Increased GSK-3-mediated synaptic deconsolidation has been suggested to be an important factor contributing to reduced spine density in mood disorders (74). Additionally, levels of activated GSK-3 are increased in postmortem brain tissue from individuals with unipolar and bipolar depression (74). In addition to BDNF, GSK-3 is deactivated by signals originating from numerous signaling pathways demonstrated to be dysregulated in mood disorders (e.g., Wnt and PI3K pathways), and is either the direct or downstream target of many mood stabilizer and antidepressant medications (75). GSK-3 activity is modulated by serotonin and dopamine, and is a critical node at the intersection of multiple neurotransmitter and cell signaling cascades (68). As a result, GSK-3 modulates not only synaptic plasticity but also apoptotic mechanisms and, in turn, plays a critical role in mediating cellular resilience (75). For this reason, GSK-3 has received much attention for its potential to be targeted as an early intervention strategy during the prodrome period.

#### **IDENTIFYING IMPAIRED CELLULAR RESILIENCE**

Stress, allostatic overload, and neuroinflammation function together to impair synaptic plasticity and cellular resilience. Disrupted plasticity along with increased cellular vulnerability contributes significantly to the pathophysiology of mood disorders and directly to the neuroprogressive nature of the disease course (3, 76). Some of the key mechanisms of disease progression affecting cellular resilience include: oxidative stress, decreased neurotrophic factor expression, reduced neurogenesis, impaired regulation of calcium, altered endoplasmic reticulum and mitochondrial function, together with dysregulated energy metabolism and insulin signaling. Each of these mechanisms are mediated by allostatic overload and neuroinflammation [for detailed reviews see Ref. (3, 36, 76–78)]. Together, these processes demonstrate that in addition to synaptic integrity, maintenance of cellular homeostasis is critical for facilitating cellular resilience and attenuating mood disorder pathogenesis (79), which is also likely to enhance the capacity for treatment response during later stages of the disorder (80).

Cellular vulnerability and resilience are mediated by apoptotic and anti-apoptotic intracellular signaling cascades, respectively. Apoptosis is important for the regulation of developmental processes and prevention of cancerous growths. Excessive apoptosis in neuronal systems, however, leads to neurodegeneration and certain cell populations are at increased risk of stress-mediated apoptotic cell death (80). Apoptosis is a tightly regulated and energy-dependent process, which coordinates programed cell death in response to different stimuli (81). This can occur through stimulation of death receptor proteins [i.e., tumor necrosis factor (TNF) receptor] by cytokines of the TNF superfamily or in response to mitochondrial degradation. These stimuli result in activation of executioner caspases that function to coordinate cellular process necessary for apoptosis, including cessation of cell repair processes and cell cycle progression, cytoskeletal and nuclear disassembly, and flagging the cell for phagocytosis (82). Distinct classes of antidepressants and mood stabilizers have been demonstrated to facilitate cellular resilience to prevent progression of pro-apoptotic processes, and novel treatments are currently being developed to target these specific mechanisms (83). Biomarkers that characterize the level of neuronal vulnerability relative to resilience may prove useful as biomarkers of prodromal mood disorder pathology. This has been demonstrated for later stages of bipolar disorder (84), however more studies are needed to determine the utility of such cell danger biomarkers during the mood disorder prodrome (22).

## **OPPORTUNITIES FOR PREVENTION AND INTERVENTION**

#### **IDENTIFYING VULNERABILITIES AND BUILDING RESILIENCE AT THE CELLULAR LEVEL**

Identification of individuals at risk of developing a mood disorder, or those in the prodromal stage, provides a potential opportunity to target these mechanisms for neuroprotective interventions that enhance cellular resilience, maintain synaptic plasticity and boost psychological resilience (**Figure 1**) (85). One of the longest held notions of brain plasticity is that certain critical periods or windows exist in development, during which circuitry is consolidated for lifetime functionality. Recently, there is a rising consensus that developmentally induced plasticity can, to an extent, be reversed by "re-opening" those windows of plasticity (86). Hyman and Nestler (87) have underscored the importance of shifting the brain into an "adaptive state" to necessitate the antidepressant response. Their theory of "initiation and adaption" is exemplified by psychotropic drugs wherein primary molecular targets that initiate alterations in brain function activate homeostatic mechanisms that return the system to an adaptive and treatment responsive state (87). Plasticity and cellular resilience are thus necessary for the efficacy of antidepressants and mood stabilizing treatments. McGorry and colleagues (6, 88) and others (89) have demonstrated this concept with pre-psychotic interventions, and repeatedly emphasized the need to take advantage of the "windows of opportunity" present within the prodromal stages of psychiatric disease (6, 88, 89). During this stage, the course of the disease remains theoretically plastic and amenable to intervention (90). Previous literature indicates that once risk or prodromal symptoms of mood disorders are identified, there is some (91), but not unequivocal (92) evidence that early intervention in adolescents can significantly reduce mood-related symptoms and incidence of fully diagnosable psychiatric disorders such as depression (93– 95). Neuroprotective pharmacotherapies together with appropriate psychotherapy may reduce the risk of neuropsychiatric disease progression in young people which, together with allostatic load reducing behavioral interventions, may significantly slow the trajectory of the disease course into adulthood (6, 36, 96). Such interventions may include reducing lifestyle mediators of allostatic load (19, 97).

**FIGURE 1 | A representation of the conceptual balance between vulnerability and resilience in prodromal individuals**. The scale's balance beam teeters between vulnerability and resilience as scale pans are loaded with different positive and negative biological, psychological and social factors. The presence or absence of these factors influence the ability of the individual to cope with stressors, and maintain allostasis. **(A)** Prodromal individuals are somewhat predisposed to vulnerability; but with intervention **(B)** an individual may adopt more adaptive environmental coping strategies, support mechanisms, general healthy lifestyle choices, and/or receive pharmacological interventions that collectively enhance physiological and psychological resilience.

#### **COGNITIVE AND BEHAVIORAL INTERVENTIONS TO BUFFER STRESS AND BUILD RESILIENCE**

Individuals provided with effective social and emotional support to help cope with stressors that are adverse and potentially taxing will be much better placed to limit associated biological costs and maintain allostasis (98). The absence of emotional or social support and the implementation of maladaptive coping strategies can enhance the toxic effects of stress and contribute to allostatic overload (98). Exposure to regular and controllable stressors over the course of childhood and adolescence is essential for the development of effective coping strategies. Through such exposure, an individual can develop a repertoire of these coping strategies. Mathew and Nanoo (99) found that adaptive coping strategies (e.g., employing self-control, accepting responsibilities, problem solving, seeking social support, or positive reappraisal) are protective for suicide risk in adolescents. Conversely, maladaptive coping strategies, such as confrontation, distancing, and escape-avoidance were reported to be significant risk factors associated with adolescent suicide attempts (99). These findings provide evidence to support the notion that coping strategies can act as protective factors against both the development and progression of mood disorders. Importantly, educating children and adolescents in protective coping skills may be a promising intervention that could be implemented as early as elementary school. In recent years, patterns of threat perception such as optimism have attracted much attention in relation to later mood, coping, and immune change in response to stress (100, 101). Moreover, it has been found to be protective against the development of depressive symptoms in later life (102). Its potential role in buffering against the negative emotional consequence of adverse events has led to a view of optimism as an index of resilience (103). Optimists may also choose lifestyles that promote physical as well as mental health, thereby reducing other aspects of allostatic load.

Healthy lifestyle, similar to optimism, provides a solid foundation for adaptation, and increases available resources for buffering the neurodegenerative effects of stress. Specifically, previous literature highlights the importance of healthy diet, adequate sleep, avoidance of smoking, and sufficient exercise (104). A populationbased study reported higher emotional well-being among physically active youths, independent of social class and health status (105). Across a 2-year period, Motl and colleagues (106) found changes in physical activity were inversely related to a change in depressive symptoms. Levels of physical activity in childhood can modulate the risk of adult depression (107). Exercise modulates many of the core biomarkers of neuroprogression, including inflammation, oxidative stress, and neurotrophins (108). Poor eating habits and sleep have been linked to the manifestation of toxic stress and unhealthy growth in pediatrics by disrupting the architecture of the plastic, adaptive brain (109). There is now extensive evidence that poor diet quality is a risk for adolescent depression (110), and new data suggests that maternal diet influences the mental health of offspring (111). Similarly, smoking increases the risk of mood and anxiety disorders, and appears to influence similar biological pathways (112, 113). Parents and care givers of younger children need to be informed of the potential impact that a healthy lifestyle can have in mitigating mood-related symptoms and problematic behaviors. Low-risk interventions such as those aforementioned are critical for enhancing both psychological and biological resilience to stress. When such perspectives and lifestyle health behaviors are consolidated early in childhood and adolescence, the cumulative effect may be meaningful (103).

#### **CONCLUSION**

Early intervention offers the possibility of altering the trajectory of mood disorder pathology. In so doing, we may curtail the progressive nature of the illness, both through neuroprotection and maintenance of peripheral health. Prevention and intervention treatments should go beyond stabilizing mood to include various and complementary strategies for reducing allostatic load, perhaps through psychoeducation and lifestyle-related interventions, including effective stress management. The combination of these techniques with specific pharmacotherapies may significantly improve functional outcomes by both reducing cellular insults and enhancing resilience. In so doing, this optimizes the capacity for maintenance of synaptic integrity and cellular resilience, which must be aggressively targeted as a therapeutic strategy during the prodromal stage of mood disorder pathology (90). This neuroprotective approach not only slows neuroprogression associated with the disease, but lays a foundation for more treatment-responsive outcomes during later stages.

#### **AUTHOR CONTRIBUTIONS**

Adam J. Walker, Yesul Kim, J. Blair Price, Rajas P. Kale, Jane A. McGillivray, Michael Berk, and Susannah J. Tye each made contributions to the writing of this manuscript.

#### **ACKNOWLEDGMENTS**

This work was supported by a Mayo Minnesota Partnership grant to Susannah J. Tye. Adam J. Walker is supported by an Australian Postgraduate Award. Yesul Kim is supported by a Deakin University Award. Michael Berk is supported by a NHMRC Senior Principal Research Fellowship 1059660.

#### **REFERENCES**

of depressive symptoms in adolescents. *J Am Acad Child Adolesc Psychiatry* (2004) **43**(3):298–306. doi:10.1097/00004583-200403000-00011

5 years: a prospective cohort study. *J Am Acad Child Adolesc Psychiatry* (2013) **52**(10):1038–47. doi:10.1016/j.jaac.2013.07.002

**Conflict of Interest Statement:** Michael Berk has received grant/research support from the NIH, Cooperative Research Centre, Simons Autism Foundation, Cancer Council of Victoria, Stanley Medical Research Foundation, MBF, NHMRC, Beyond Blue, Rotary Health, Geelong Medical Research Foundation, Bristol Myers Squibb, Eli Lilly, GlaxoSmithKline, Meat and Livestock Board, Organon, Novartis, Mayne Pharma, Servier and Woolworths, has been a speaker for Astra Zeneca, Bristol Myers Squibb, Eli Lilly, GlaxoSmithKline, Janssen Cilag, Lundbeck, Merck, Pfizer, Sanofi Synthelabo, Servier, Solvay, and Wyeth, and served as a consultant to Astra Zeneca, Bristol Myers Squibb, Eli Lilly, GlaxoSmithKline, Janssen Cilag, Lundbeck, Merck, and Servier. The other authors have no conflicts to report.

*Received: 29 October 2013; accepted: 17 March 2014; published online: 09 April 2014. Citation: Walker AJ, Kim Y, Price JB, Kale RP, McGillivray JA, Berk M and Tye SJ (2014) Stress, inflammation, and cellular vulnerability during early stages of affective disorders: biomarker strategies and opportunities for prevention and intervention. Front. Psychiatry 5:34. doi: 10.3389/fpsyt.2014.00034*

*This article was submitted to Neuropsychiatric Imaging and Stimulation, a section of the journal Frontiers in Psychiatry.*

*Copyright © 2014 Walker, Kim, Price, Kale, McGillivray, Berk and Tye. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Neural responses during social and self-knowledge tasks in bulimia nervosa

### **Carrie J. McAdams 1\* and Daniel C. Krawczyk 1,2**

1 Department of Psychiatry, The University of Texas Southwestern Medical Center, Dallas, TX, USA

2 School of Behavioral and Brain Sciences, Center for Brain Health, The University of Texas at Dallas, Dallas, TX, USA

#### **Edited by:**

Paul Croarkin, Mayo Clinic, USA

#### **Reviewed by:**

Peter G. Enticott, Monash University, Australia Jamie Morris, University of Virginia, USA

#### **\*Correspondence:**

Carrie J. McAdams, Department of Psychiatry, The University of Texas Southwestern Medical Center, 6363 Forest Park Road BL6.110E, Dallas, TX 75390-8828, USA e-mail: carrie.mcadams@ utsouthwestern.edu

Self-evaluation closely dependent upon body shape and weight is one of the defining criteria for bulimia nervosa (BN). We studied 53 adult women, 17 with BN, 18 with a recent history of anorexia nervosa (AN), and 18 healthy comparison women, using three different fMRI tasks that required thinking about self-knowledge and social interactions: the Social Identity task, the Physical Identity task, and the Social Attribution task. Previously, we identified regions of interest (ROI) in the same tasks using whole-brain voxel-wise comparisons of the healthy comparison women and women with a recent history of AN. Here, we report on the neural activations in those ROIs in subjects with BN. In the Social Attribution task, we examined activity in the right temporoparietal junction (RTPJ), an area frequently associated with mentalization. In the Social Identity task, we examined activity in the precuneus (PreC) and dorsal anterior cingulate (dACC). In the Physical Identity task, we examined activity in a ventral region of the dACC. Interestingly, in all tested regions, the average activation in subjects with bulimia was more than the average activation levels seen in the subjects with a history of anorexia but less than that seen in healthy subjects. In three regions, the RTPJ, the PreC, and the dACC, group responses in the subjects with bulimia were significantly different from healthy subjects but not subjects with anorexia. The neural activations of people with BN performing fMRI tasks engaging social processing are more similar to people with AN than healthy people.This suggests biological measures of social processes may be helpful in characterizing individuals with eating disorders.

**Keywords: mentalization, identity, theory of mind, eating disorders, anorexia, bulimia, neuroimaging, social behavior**

#### **INTRODUCTION**

Bulimia nervosa (BN) is an eating disorder characterized by frequent binge-eating followed by purging behaviors in concert with a self-esteem that is overly associated with body shape and weight (1). The symptoms of many eating disorder patients change during their lives (2, 3). For example, a patient may develop restricting behaviors with weight loss in high school, begin binging and purging behaviors at a low weight, continue binge-purge behaviors at a healthy weight throughout college, and then cease the purging behaviors but have occasional binge-eating problems.  This diagnostic instability makes clinical treatment as well as research into eating disorders challenging (4, 5). A better understanding of biological and cognitive similarities and differences that contribute to eating disorders may improve clinical treatment. Currently, treatment of BN leads to sustained recovery in only about half of the patients (6, 7). Through the use of fMRI, we examined neural activations related to social processes in BN.

A specific set of neural regions is modulated in response to tasks that require thinking about people in healthy subjects (8, 9); this provides a framework to assess differences related to psychiatric illnesses. Severe impairments in social interaction are one of the diagnostic criteria for autistic spectrum disorders (1), but problems in social cognition have been reported in many psychiatric illnesses (10–14). Decreased social cognition has been reported in a variety of behavioral tasks in adults with AN (15–17). In BN, recent studies have concluded that there was little evidence of social cognition differences in psychological tasks (18, 19), although far fewer studies of social cognition have been completed in BN than in AN.

Because both BN and AN include in their diagnostic criteria an association between appearance and self-esteem (1), these experiments focused on neural pathways related to thinking about oneself. Self-esteem is a term used to describe one's overall sense of one's own value as a person, and is generally considered a fairly stable psychological characteristic (20). Although the diagnostic criteria in eating disorders connect self-esteem specifically to physical appearance, similarly unrealistic social expectations are reported and observed in eating disorder patients (21, 22). Selfknowledge, as used in MRI tasks, relates to the ability to evaluate oneself, and is expected to be a process that involves self-esteem as well as other criteria. For example, an individual whose self-esteem is highly related to appearance might be very good at her work, and correctly describe herself as a competent employe, but maintain an overall low self-esteem because of perceived inadequacies of appearance. Furthermore, low self-esteem has been related to

prognosis in AN (23,24) as well as onset of bulimic symptoms (25). Negative beliefs about one's self, unrelated to physical appearance, have been observed in eating disorders (26, 27), and neural differences in the processing of these negative self-beliefs have been seen in BN (28). These data show not only that psychological similarities in self-esteem are present in AN and BN, but also that self-esteem is an important factor in assessing the prognosis and severity of eating disorders.