Accepted Manuscript

Amygdala activation and connectivity to emotional processing distinguishes

asymptomatic patients with bipolar disorders and unipolar depression

Mayuresh S. Korgaonkar, May Erlinger, Isabella A. Breukelaar, Philip Boyce, Philip

Hazell, Cassandra Antees, Sheryl Foster, Stuart M. Grieve, Lavier Gomes, Leanne M.
Williams, Anthony W.F. Harris, Gin S. Malhi
PII: S2451-9022(18)30236-2
DOI: 10.1016/j.bpsc.2018.08.012
Reference: BPSC 328

To appear in: Biological Psychiatry: Cognitive Neuroscience and

Neuroimaging

Received Date: 1 June 2018

Revised Date: 6 August 2018
Accepted Date: 18 August 2018

Please cite this article as: Korgaonkar M.S., Erlinger M., Breukelaar I.A., Boyce P., Hazell P., Antees
C., Foster S., Grieve S.M., Gomes L., Williams L.M., Harris A.W.F. & Malhi G.S., Amygdala activation
and connectivity to emotional processing distinguishes asymptomatic patients with bipolar disorders
and unipolar depression, Biological Psychiatry: Cognitive Neuroscience and Neuroimaging (2018), doi:
10.1016/j.bpsc.2018.08.012.

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Amygdala activation and connectivity to emotional processing distinguishes asymptomatic

patients with bipolar disorders and unipolar depression.

Short Title: Emotion processing in bipolar and unipolar depression

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Mayuresh S. Korgaonkar1,2, May Erlinger1, Isabella A. Breukelaar1, Philip Boyce2, Philip Hazell2,

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Cassandra Antees1, Sheryl Foster3,4, Stuart M. Grieve5,6, Lavier Gomes3, Leanne M. Williams1,7,8,

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*Anthony W.F. Harris1,2, *Gin S. Malhi2,9

*co-senior authors

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1
Brain Dynamics Centre, Westmead Institute for Medical Research, The University of Sydney,

Westmead, NSW, Australia

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2
Discipline of Psychiatry, Sydney Medical School, The University of Sydney, NSW, Australia
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3
Department of Radiology, Westmead Hospital, Westmead, NSW, Australia
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4
Discipline of Medical Radiation Sciences, Faculty of Health Science, The University of Sydney,

NSW, Australia
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5
Sydney Translational Imaging Laboratory, Heart Research Institute, Charles Perkins Centre and

Sydney Medical School, The University of Sydney, NSW, Australia

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6
Department of Radiology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia
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7
Psychiatry and Behavioral Sciences, Stanford University, Stanford, CA, USA
8
Sierra-Pacific Mental Illness Research, Education, and Clinical Center (MIRECC), Palo Alto VA,

Palo Alto, CA, USA

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CADE Clinic, Department of Psychiatry, Royal North Shore Hospital, Sydney, NSW, Australia

Correspondence addressed to:

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Dr. Mayuresh Korgaonkar (M.Korgaonkar@sydney.edu.au,)

Brain Dynamics Centre,

Westmead Institute for Medical Research,

176 Hawkesbury Road,

Westmead, NSW 2145, Australia

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T: +61 02 86273301

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Keywords: bipolar disorders, depression, remission, emotion processing, fMRI connectivity,

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amygdala

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Word count:
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Abstract: 250 words

Text: 4000 words

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Number of figures: 3
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Number of tables: 4
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Supplementary Information: Supplementary Methods & Results

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ABSTRACT

Background: Mechanistically based neural markers such as amygdala reactivity offer one

approach to addressing the challenges of differentiating bipolar and unipolar depressive disorders,

independently from mood state and acute symptoms. Although emotion-elicited amygdala

reactivity has been found to distinguish bipolar from unipolar patients in depressed states, it

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remains unknown whether this distinction is trait-like and present in the absence of an acutely

depressed mood. We address this gap by investigating bipolar (BP) and unipolar major depressive

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disorder (MDD) populations in remission.

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Methods: 73 participants (23 BP and 25 MDD patients matched for age-gender, number of

depressive episodes and severity; 25 age-gender matched healthy individuals) completed supra-

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and subliminal processing of threat, sad, happy and neutral faces during fMRI. We compared
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groups for activation and connectivity for the amygdala.

Results: BP participants had lower left amygdala activation than MDD during supraliminal and
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subliminal threat, sad and neutral processing and for subliminal happy faces. BP participants also
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exhibited lower amygdala connectivity to the insula and hippocampus for threat and to medial
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orbito-frontal for happy supraliminal and subliminal processing. BP participants also demonstrated

greater amygdala-insula connectivity for sad supraliminal and subliminal face processing. Both
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patient groups were distinct from controls across several measures for activation and connectivity.

Conclusion: Independent of valence or level of emotional awareness amygdala activation and

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connectivity during facial emotion processing can distinguish BP and MDD patients. These
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findings provide evidence that this neural substrate could be a potential trait-marker to

differentiate these two disorders largely independent of illness state.

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INTRODUCTION

Approximately 60% of patients with bipolar disorder are initially misdiagnosed as major

depressive disorder (1, 2). This is because bipolar disorder often first presents in the depressive

phase of the illness and bipolar depression is similar to unipolar major depression in terms of

clinical symptoms. This can result in a considerable delay in diagnosis of up to a decade, and

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during this time patients may be subjected to suboptimal treatment with predictably poorer

outcomes – compounding the societal and economic burden of this disorder (3, 4). It is

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increasingly evident that existing diagnostic clinical tools are incapable of differentiating the two

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disorders in their early stages – prior to the development of mania (5-7). Clearly, identifying

pathophysiological markers that could reliably differentiate these two disorders would therefore

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have substantial clinical value.
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Both bipolar and major depressive disorders exhibit abnormalities of emotion processing with
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consistent abnormal activation in the amygdala (8-10). Amygdala responsivity during emotion
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processing could be a core feature in distinguishing bipolar depression from major depression.
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Prior studies suggest that both abnormal activation and functional connectivity of the amygdala

elicited by negative and positive facial emotions may be a state-like marker for bipolar disorder
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(11-14). The focus of these studies has been on the comparison of depressed bipolar individuals

relative to those with major depression (11-13), and bipolar individuals in depressed versus
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remitted states (13, 14). However, the directionality of effects across these studies have largely
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been inconsistent. These differences are evident but may be dependent on mood state, in particular

to depression. It remains unknown whether such discrimination at a neural level persists in

remitted states and could potentially serve as a trait marker for differentiating these disorders

irrespective of whether they are depressed or not. Such a marker could help better understand the

pathophysiology underlying both these disorders, identify risk factors for developing these

disorders and potentially enable clarity in diagnosis early from onset. Notably, the numbers of
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studies directly comparing bipolar and major depressive disorders are few, and no previous study

has compared patients in euthymic or remitted states.

Amygdala responsivity elicited under conditions of both bottom-up automatic reactivity and top-

down evaluation is a promising probe for addressing this gap in knowledge (15). Previous work

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has shown that major depression is characterized by abnormal amygdala responses during both the

automatic reactivity to emotion stimuli (when presented subliminally) and during the more

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controlled evaluation of these stimuli (when presented supraliminally) (16, 17). Amygdala

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responsivity probed by subliminal emotion stimuli has also been shown to distinguish bipolar from

major depressive disorders in a depressed state, with bipolar individuals exhibiting greater

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amygdala activation for happy whereas lower amygdala activation for sad (18).
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In this study, we examined amygdala responsivity and connectivity in a cohort of remitted bipolar
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and unipolar depressed individuals. To determine whether stimulus valence or level of awareness
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in processing modulates disease-related activity within the emotion processing network, we

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employed two fMRI tasks that tapped into both supraliminal and subliminal processing of

emotions comprising threat, sadness, happiness and neutral. Based on previous studies comparing
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bipolar and unipolar patients in the depressed state, we hypothesized amygdala activity and

connectivity in response to emotion processing elicited by a range of facial emotions to

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differentiate the two disorders in remission.

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METHODS AND MATERIALS

Participants

The study involved 81 individuals (aged 18-60): 31 remitted individuals with Bipolar I disorder

[BP], 25 remitted individuals with major depressive disorders [MDD] and 25 healthy control
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individuals [HC]. Participants underwent functional MRI scans and testing at the Brain Dynamics

Centre, Westmead Institute for Medical Research.

Study protocol

BP and MDD patients were recruited through general practitioner referrals and clinics in the

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Western Sydney Local Health District and in the greater Sydney area or through community

advertisements. Patients met DSM-IV criteria for bipolar I or MDD and were required to be

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remitted at time of testing, screened through a series of clinical assessments, including the

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Structured Clinical Interview for DSM-IV Axis I Disorders (19), Mini-International

Neuropsychiatric Interview (20), Hamilton Rating Scale for Depression (HDRS17) (21), and

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Depression Anxiety and Stress Scale (DASS) (22). Remission was defined as at least 14 days of
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being asymptomatic i.e. no manic episode and a depression severity score of HRSD17 < 7 for at

least 2 weeks prior to and at the time of testing. BP patients were currently taking lithium (n=15),
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anti-psychotics (n=18), antidepressants (n=6), anti-convulsant (n=9) or benzodiazepine (n=2),

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whereas all MDD patients were on antidepressants (13 SSRIs and 12 on SNRIs) at time of testing.
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Healthy participants were recruited from the general community and screened extensively using

the MINI to ensure they did not meet DSM-IV criteria for any recurrent or non-recurrent Axis I or
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other psychiatric disorders. All participants were required to have no previous history of

psychiatric diagnoses (other than BP or MDD for the respective patient groups), current or
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previous substance dependence, history of brain injury, previous loss of consciousness greater than
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ten minutes, self-reported serious medical conditions, or any contraindications for a MRI scan.

Eight BP participants had to be excluded (one was manic during testing, four did not finish both

emotional processing tasks addressed in this study and three due to excessive motion during the

tasks), resulting in total 23 BP, 25 MDD and 25 HC individuals for analyses.

All participants provided informed consent and the study was conducted in accordance with the

ethical guidelines of the institutional review board.

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fMRI acquisition and task details

MRI data was acquired on a 3T GE Signa HDx scanner (GE Healthcare, Wisconsin) at Department

of Radiology, Westmead Hospital using an 8-channel head coil. Functional MR images were

acquired using echo planar imaging (TR/TE=2500/27.5ms, matrix=64x64, FOV=24cm, flip

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angle=90°). Forty slices, each 3.5mm thick, covered the whole brain in each volume. For each

task, 120 volumes were collected with a total scan time of 5min and 8s. Specifically, the scan

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consisted of five tasks (only two of which are reported and analyzed in this paper; supraliminal

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and subliminal emotional processing) and a 3D T1-weighted structural MRI scan

(TR/TE=8.3/3.2ms; flip angle=11°; TI=500ms; NEX=1; ASSET=1.5; Frequency direction: S/I;

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matrix=256x256; 180 contiguous 1mm sagittal slices covering the whole brain; 1mm3 voxels).
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The details of the emotional processing tasks have been previously described (17, 23). Briefly, in

the supraliminal emotional processing task participants passively viewed a series of emotional
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faces (angry, disgusted, fearful, sad, happy, and neutral) for 500ms per stimulus, with an
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interstimulus interval (ISI) of 750ms. In the subliminal task, participants were presented with
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identical faces; however, the stimulus duration was 10ms, and was immediately followed with a

neutral face for 150ms. To control for conscious detection on the basis of perceptual features, the
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neutral masked faces were offset by 1 degree in random directions. Prior testing using behavioral

psychophysiological testing indicates that presentation of facial stimuli at ≤ 20ms meets signal
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detection criteria for discrimination of emotional expression (24). The ISI within the subliminal
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task was 1100ms, to ensure all stimulus presentation was equivalent across conditions and task.

Across both tasks there were a total of 240 stimuli, grouped into blocks of 8 faces of the same

emotion, and repeated 5 times. In both tasks, participants were instructed to pay attention to each

emotion face in order to respond to post-test questions.

fMRI data analyses

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Data was pre-processed and analysed using SPM8 (http://www.fil.oin.ucl.ac.uk/spm) and has been

described previously (23) and in detail in the supplementary section. Briefly, data was first

realigned and unwarped, screened for motion artifacts using the Artifact Detection Tools

(www.nitrc.org/projects/artifact_detect/), normalization parameters to transfer to standard space

were estimated, global signal from CSF/white matter was removed, followed by smoothing and

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high pass filtering. First-level general linear models for each task consisted of regressors

representing blood oxygen level dependent (BOLD) responses for each emotion block and the

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motion parameters, and were used to derive the following contrast images: for threat-related

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emotions (anger, fear, disgust) vs baseline average (to assess activation and connectivity elicited

by signals relevant to direct threat, indirect threat and threat of contaminants), sad vs baseline (to

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assess activation and connectivity elicited by signals of potential loss), happy vs baseline (to assess
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activation and connectivity elicited by signals of potential positive affect and reward), and neutral

vs baseline (to assess activation and connectivity elicited to a neutral condition). We chose to
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evaluate each emotion relative to baseline average (i.e. implicit baseline) as compared to neutral
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because of recent mounting evidence that neutral faces may not be suitable baseline comparisons
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(25). However, for comparisons with previous fMRI studies of emotion processing using neutral

as baseline, we also evaluated contrasts of each emotion relative to neutral in supplementary

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analyses. All contrast images were individually normalized to standard space using the warps

estimated from the pre-processing steps noted previously and these normalized contrast maps were
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then used for all second-level analyses.

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Activation analysis

The primary second-level analysis for both supraliminal and subliminal emotional processing tasks

consisted of voxel-wise analyses of variance (ANOVAs) between group (2: BP and MDD) by

emotion (6: Anger, Fear, Disgust, Sad, Happy, Neutral) to compare neural activations between the

BP and MDD groups. These were conducted in the left and right amygdala regions of interest
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(ROI) defined using the AAL (automated anatomical labeling) atlas (we also performed

exploratory whole brain analyses, reported in the supplementary section). Post-hoc voxel-wise

analyses for individual emotion contrasts were performed for significant main effects. To control

for Type 1 errors, a cluster level false discovery rate (FDR) correction procedure was employed. A

corrected alpha level of 0.05 was estimated using Monte Carlo simulations using REST AlphaSim

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software for an uncorrected height threshold of 0.05. For the bilateral amygdala ROI, the

minimum significant cluster size to maintain the FDR threshold was 27 voxels. Identical voxel-

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wise analyses were also run between patient groups and healthy controls (reported in the

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supplementary section).

Connectivity analysis

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Functional connectivity analyses (generalized psychophysiological interactions: gPPI) (26) were

performed using the left amygdala as the seed region, selected based on the group-by-emotion
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activation findings. The details for the gPPI model and analysis are described in the supplementary

section. As done for the activation analyses, connectivity maps were compared voxel-wise in a
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second level analysis between the groups using FDR corrections. We evaluated the threat, sad and
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neutral emotions in a single ANOVA and happy emotion contrasts in separate second level ROI
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analyses as we tested connectivity differences between groups in specific networks depending on

emotion (27). The negative affect network (comprising of the subgenual [sgACC] and pregenual
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anterior cingulate cortex [pgACC], bilateral hippocampus, insula, and amygdala) was used for
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connectivity analyses of the threat, sad, and neutral emotion contrasts. The positive affect network

(comprising of the dorsal anterior cingulate cortex [dACC], medial orbitofrontal cortex [mOFC],

bilateral caudate, putamen, and pallidum) was used for the connectivity analysis of the happy

emotion contrast. These networks were chosen for their relevance to the neural circuitry of

negative and positive emotional processing (27) and ROIs defined using AAL atlas and meta-

analyses (28). For the negative affect network, the minimum significant cluster size to maintain a
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FDR corrected alpha 0.05 threshold was 71 voxels, and for the positive affect network, was 80

voxels.

Correlation with symptoms

Beta values (averaged) were extracted from the significant activation and connectivity clusters

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found between BP and MDD groups and were tested for correlations with symptom severity

(HRSD17, DASS, young mania rating (YMRS) scores) using SPSS. We examined correlations

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within each group separately as well as partial correlations with the pooled patient group

controlling for diagnostic category.

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Finally, we also evaluated accuracy of the significant activation and connectivity clusters in

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classifying the patient groups using backwards stepwise (Wald) binary logistic regression analyses
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and tested generalization of these models using leave-one-out cross-validation statistics.
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RESULTS
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Table 1 shows the demographic and clinical characteristics for all participants. BP patients were

matched to the MDD group for age, gender, number of depressive episodes, and depression
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severity (based on HRSD17 and DASS scores). The BP and MDD groups were also matched to

controls for age and gender but had higher DASS depression scores than controls (p<0.05 for two
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group comparisons). For fMRI comparisons for each patient group relative to controls, we tested
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in a post-hoc analysis if effects also survived controlling for the DASS measures (see

supplementary analyses).

INSERT TABLE 1 HERE

Activation results

Supraliminal faces task

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BP were found to have significant hypoactivation relative to MDD in the left amygdala

(significant main effect of group across all emotions). This effect was significant for threat (main

effect of group across threat emotions), sad and neutral faces (Table 2; Figure 1A). There were no

significant differences in the amygdala for happy faces. When we unpacked the main effect for

threat, this profile of relative hypoactivation for BP versus MDD was apparent for the individual

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emotional expressions of disgust and fear (details in Supplementary Results). Neither of the patient

groups differed significantly from controls for supraliminal face processing.

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FIGURE 1 ABOUT HERE

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TABLE 2 ABOUT HERE

Subliminal faces task

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Similar to supraliminal processing, BP participants had a significant hypoactivation in the left
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amygdala when compared to MDD participants (significant main effect of group across emotions).

In post-hoc comparisons, this effect was consistently significant for each of the individual
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emotions (Table 2, Figure 1B): main effect of group for threat faces and for the individual threat
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emotions (anger, fear, disgust), sad, neutral, and happy faces. In contrast to the supraliminal
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processing, BP participants had significantly reduced amygdala activation across all emotions

whereas MDD participants had greater activation only in the threat emotions relative to controls.
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Connectivity results
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We evaluated connectivity related to only the left amygdala based on the activation findings in this
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region.

Supraliminal faces task

For the negative affect network, there was a significant main effect of group for connectivity of the

left amygdala with the sgACC, right amygdala and right hippocampus and a significant group-by-

negative emotions interaction for connectivity with the insula (Table 3; Figure 2A). Similarly, for

the threat emotions, a main effect of group and group-by-emotion interaction was also observed
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for the right hippocampus and insula respectively. However, between group differences in

connectivity to sgACC for any emotion and to any of the regions for the individual threat emotions

did not meet the significance threshold in post-hoc comparisons (supplementary results). For the

other emotions, BP group had relatively lower amygdala-hippocampal connectivity for sad and

neutral faces and lower left-right amygdala connectivity for neutral faces in comparison to both

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MDD and healthy controls. MDD were also greater than controls for amygdala-hippocampal

connectivity for sad faces. In contrast, amygdala-insula connectivity for sad faces was relatively

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greater in BP than MDD but both groups were not distinct from controls.

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For the positive affect network, the BP group had lower left amygdala connectivity than MDD

with the mOFC, the left putamen, and the left caudate for happy faces (Table 3; Figure 2B). As

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compared to controls, the BP group also had significantly lower amygdala connectivity to the left
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putamen, whereas MDD had greater amygdala-mOFC connectivity relative to controls.

TABLE 3 ABOUT HERE

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FIGURE 2 & 3 ABOUT HERE

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Subliminal faces task

For the negative affect network, there was a significant main effect of group and group-by-
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negative emotion interactions for connectivity to the pgACC, bilateral insula and right
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hippocampus (Table 3; Figure 3A). Only group main effects were also observed for the sgACC
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and right amygdala. Similarly, main group and interaction effects for threat emotions were

significant for the right hippocampus and insula. Post-hoc analyses revealed that the BP group had

amygdala hypo-connectivity to insula and hippocampus mainly to anger and disgust threat

emotions relative to MDD (supplementary results). This amygdala-insula and amygdala-

hippocampal hypo-connectivity for threat also distinguished BP from controls whereas MDD were

similar to controls. BP also had lower left-right amygdala connectivity for sad faces and lower

amygdala-hippocampus connectivity for neutral faces compared to MDD. Similar to the

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supraliminal sad faces, amygdala-insula connectivity for subliminal sad faces was relatively

greater in BP than MDD. Connectivity for sad and neutral faces did not distinguish either of the

patient groups from controls.

For the positive affect network, the BP group had lower left amygdala connectivity than MDD

with the mOFC consistent with that observed for supraliminal emotional processing (Table 3;

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Figure 3B). However, both patient groups were similar in connectivity relative to controls.

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Correlation with symptoms

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Correlational analyses between mean beta weights extracted from the significant activation and

connectivity clusters with HDRS17, DASS, and YMRS symptom scores were not significant in

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either BP or MDD patient groups or the pooled patient group controlling for diagnostic
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categorization.
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Diagnostic classification analyses

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To determine whether group differences in activation and connectivity could predict patient
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diagnoses, we ran three backwards stepwise (Wald) binary logistic regression analyses: two using

the significant supraliminal and subliminal results (activation and connectivity) separately, and in
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the third regression only the significant supra- and subliminal regions from the first two backwards

stepwise regressions. All three models had a cross-validated accuracy greater than 80% (Table 4).
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The supraliminal model comprising of left amygdala activation for fear and left amygdala
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connectivity to mOFC and left caudate for happy, to the right amygdala for neutral, and to right

insula for sad emotions, had the highest classification accuracy of 88.6% (86.4% sensitivity;

90.9% specificity).

DISCUSSION
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This study demonstrates that amygdala functional activation and connectivity differ between

bipolar disorder and major depressive disorder in remission. BP participants had a profile of hypo-

activation in the left amygdala relative to MDD individuals that was consistent across level of

awareness in emotion processing (i.e. at both supraliminal and subliminal) and type of facial

emotions. Relative to MDD, BP participants also exhibited lower amygdala-insula and amygdala-

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hippocampal connectivity for the threat emotions and greater amygdala-insula connectivity for the

sad emotions for both subliminal and supraliminal processing. BP participants also had lower

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connectivity between the amygdala and the mOFC for viewing of both supra- and subliminal

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happy faces. These findings suggest that differences in amygdala activation and connectivity

during processing of facial emotions persist beyond the depressed and manic states and potentially

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could be a trait marker for differentiating these disorders.
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Clinically it is difficult to distinguish between bipolar and major depressive disorders because of
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the higher prevalence of depressive symptoms relative to hypo/manic symptoms during the course
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of bipolar disorder, and the presence of sub-threshold manic symptoms during a depressive
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episode (29). This problem has led to studies that have evaluated bipolar and unipolar patients

particularly during the depressed state (5). Emotion processing is a core problem underlying both
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these disorders and amygdala reactivity and connectivity during facial emotion processing has

been identified as a possible state marker to distinguish these disorders (11, 13, 18, 30). However,
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the directionality of effects across these studies have largely been inconsistent, with studies
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reporting greater amygdala activation in bipolar disorders for happy and fear processing (18, 30),

lower activation for angry emotions (11) and bi-directional effects (13, 18) as well as no

differences relative to MDD (11) for sad emotions. We observed lower left amygdala activation

for BP patients relative to MDD individuals in remission. This pattern was consistent across

different emotions and during both conscious and non-conscious levels of emotional processing.

While amygdala activity during both supra- and subliminal facial emotion processing for both BP
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and MDD cohorts in acute states are widely reported to be different relative to healthy individuals

(8, 9), there are only few and mainly inconsistent reports for asymptomatic cohorts. Euthymic BP

patients are found to have reduced amygdala activity relative to those in manic states (31), but

differences relative to healthy individuals have largely been inconsistent (8). We observed

normalized amygdala activation across different emotions only for supraliminal processing

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whereas subliminal processing abnormalities, particularly for threat emotions, persist in both

patient groups in remission.

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Remitted BP group also had lower connectivity relative to MDD to the insula and hippocampus

for negative emotions (except for sad stimuli for insula where an opposite pattern was observed)

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and to the medial OFC and striatal brain regions for the happy stimuli (only for supraliminal). The
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differential direction of connectivity related to the insula and particularly for sad versus other

emotions, may provide some insight into core differences between the two disorders. The insula is
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regarded as a region strongly underpinning processing of interoceptive states (32) with

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interconnections with regions associated with the experience of emotion (33) and to hubs of the
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default mode, cognitive control and cortico-striatal networks (34). Hence, it is crucial in the

integration of stimulus driven bottom up interoceptive signals with top down predictions to
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generate a current emotion awareness state. Clinically, depressed and bipolar individuals exhibit

heightened interoceptive awareness, which affects their ability to filter exogenous and endogenous
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stimuli for adaptive regulation e.g. increase in negative self-focused thought or rumination in
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depressed individuals that impairs shifting from internally focused to externally focused attention;

or an increase in energy in bipolar individuals that prevents regulating from rewarding or

threatening cues. This inability for top-down regulation might be reflective of the reduced

amygdala-insula connectivity observed for sad in MDD and that for other emotions in BP (which

was also distinct from controls), reflecting their respective core symptoms. Support for this

interpretation can be found in the work of Ellard and colleagues (35), who also found differences
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in resting state functional connectivity between the insula and the inferior parietal node of the

fronto-parietal executive brain network to distinguish unipolar and bipolar depressed participants

and other reports of differential resting connectivity related to both the amygdala and insula (36,

37). Further, reduced insula and amygdala volumes in individuals at ultra-high risk for the

development of psychosis who then subsequently transition to bipolar disorder as compared to

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those who do not, lends further support to the involvement of both these regions as risk trait

markers for bipolar disorder (38). Consistent with that observed in depressed vs. remitted BP

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cohorts (14), we also found reduced connectivity between the amygdala and mOFC during

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supraliminal happy stimuli for BP relative to MDD, which was characteristic in differentiating

remitted MDD but not BP from controls.

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There are a number of limitations to the study. First, the study did not involve BP or MDD patients

who were either currently depressed or with current manic or hypomanic symptoms. Studying
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patient groups across both acute and asymptomatic states are necessary to confirm trait markers.
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We did find some of our measures to be correlated with number of episodes and illness age of
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diagnosis (supplementary results) and different relative to controls, which lend some support for

trait-like characteristics. Second, we recognize that many of our patients were taking medication at
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the time of scanning and that the variety of treatments precludes us from parsing out their potential

effects – but at the same time it is important to note that these patients had all recovered or were
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asymptomatic and were established on therapy – therefore the pharmacological effects of

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medications was likely to be minimal in terms of direct effects and any alterations to emotion

regulatory processes per se. We did not observe medication effects on neural measures

differentiating the two disorders (supplementary results). However, future studies wherever

possible should focus on recently diagnosed individuals who are treatment naïve to rule out these

effects. Third, to evaluate diagnostic clinical value, the neural substrates from our study should be

tested for accuracy in classification and replication using an independent cohort. We observed
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almost 89% cross-validated (internal leave-one-out replication) accuracy (86% sensitivity; 91%

specificity) for our measures in classifying the two patient groups, which provides preliminary

support that these measures could have promising clinical value. Finally, sample sizes in each

patient group are relatively small and considering that a liberal FDR statistical threshold was used,

our results warrant replication in larger cohorts.

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In summary, our study found that amygdala reactivity and connectivity in response to emotion

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processing elicited by a range of facial emotions successfully differentiated bipolar disorder and

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major depressive disorders. This effect was mostly independent of the level of awareness in

emotion processing and type of facial emotions. Importantly, both patient groups were non-

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symptomatic suggesting that neural substrates for emotion processing may persist largely
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independent of illness state and could possibly serve as a trait marker that distinguishes these

disorders.
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ACKNOWLEDGEMENTS

This study was funded by the National Health and Medical Research Council (NHMRC) of

Australia Project Grant (APP1087560). The funders did not have any role in any aspects of the

work presented in this manuscript. SMG acknowledges the support of the Parker Hughes bequest

and the Heart Research Institute. MSK is supported by a NHMRC Career Development

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Fellowship (APP1090148) and GSM is supported by NHMRC Program Grant (APP1073041).

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FINANCIAL DISCLOSURES

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Prof Williams has received consultancy fees from BlackThorn therapeutics and scientific advisory

board fees from Psyberguide, One Mind Institute.

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Prof Harris has received consultancy fees from Janssen Australia and Lundbeck Australia. He has
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been on an advisory board for Sumitomo Dainippon Pharma. He has received payments for

educational sessions run for Janssen Australia and Lundbeck Australia.

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All other authors report no biomedical financial interests or potential conflicts of interest.
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TABLE LEGENDS

Table 1. Participant demographics and clinical characteristics.

Abbreviations: BP, Bipolar Disorder; DASS, Depression Anxiety and Stress Scale; HDRS17, 17-

item Hamilton Depression Rating Scale; MDD, Major Depressive Disorder; NS, not significant;

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YMRS, Young Mania Rating Scale.

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Table 2. Differences in amygdala activation for both supraliminal and subliminal emotional face

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processing for euthymic Bipolar Disorder (BP) & remitted Major Depressive Disorder (MDD)

groups. Effects significant at cluster level FDR correction (p<0.05) are shown. (L-left; n.s. – not

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significant)
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Table 3. Differences in left amygdala connectivity in the negative and positive affect networks for
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both supraliminal and subliminal emotional face processing for euthymic Bipolar Disorder (BP) &
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remitted Major Depressive Disorder (MDD) groups. Effects significant at cluster level FDR
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correction (p<0.05) are shown. (L-left; R-right; mOFC-medial orbitofrontal cortex; n.s. – not

significant)
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FIGURE LEGENDS

Figure 1. Amygdala activation levels during supraliminal (A) and subliminal (B) emotion face

processing for euthymic bipolar disorder (BP), remitted major depressive disorder (MDD) and

healthy control (HC) groups. Brain image shows significant amygdala activation for the main

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effect of emotion comparing the BP & MDD groups (cluster level FDR corrected p<0.05). Bars

represent mean activation for significant clusters for different emotions, and error bars represent

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standard deviations. * indicates significant group differences from the voxel wise post hoc

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analyses.

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Figure 2. Left amygdala connectivity in the negative affect network (A) and the positive affect
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network (B) during supraliminal emotion face processing for euthymic bipolar disorder (BP),

remitted major depressive disorder (MDD) and healthy control (HC) groups. Only facial emotions
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with significant connectivity differences are shown in the plot. Bars represent mean activation for
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significant clusters for different emotions, and error bars represent standard deviations. * indicates
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significant group differences. Brain image shows nodes (spheres) of the negative & positive affect

network and connections (arrows) that were significant between the BP & MDD groups.
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Figure 3. Left amygdala connectivity in the negative affect network (A) and the positive affect
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network (B) during subliminal emotion face processing for euthymic bipolar disorder (BP),
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remitted major depressive disorder (MDD) and healthy control (HC) groups. Only facial emotions

with significant connectivity differences are shown in the plot. Bars represent mean activation for

significant clusters for different emotions, and error bars represent standard deviations. * indicates

significant group differences. Brain image shows nodes (spheres) of the negative & positive affect

network and connections (arrows) that were significant between the BP & MDD groups.
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Table 1. Participant demographics and clinical characteristics

BP (n=23) MDD (n=25) HC (n=25) f/x^2 p-value

Age, y, mean ± SD (range) 33.48 ±13.43 (18 - 62) 32.83 ± 12.97 (18 -62) 34.29 ± 13.31 (18 - 60) 0.08 NS
Gender, % female 65.22 64.00 65.22 0.15 NS
0.038 (BP > HC)
DASS Dep, mean ± SD 8.82 ±7.76 7.76 ± 5.61 3.89 ± 4.38 3.45
(MDD > HC)
DASS Anx, mean ± SD 6.55 ± 7.26 5.00 ± 5.20 2.22 ± 3.08 3.03 NS
DASS Str, mean ± SD 8.00 ± 8.63 4.00 ± 4.09 6.39 ± 4.71 2.52 NS
# of depressive episodes,
16.95 ± 31.35 (1-99) 12.08 ± 15.93 ( 1- 60) n/a 0.45 NS

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mean ± SD (range)
# of Manic Episodes, mean
14.68 ± 28.37 (1-99) - -
± SD
HDRS17, mean ± SD 5.22 ± 6.11 5.6 ± 3.45 n/a 0.07 NS

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YMRS, mean ± SD 2.09 ± 2.58
Lithium, number (%) 15 (65) - -

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Anti-convulsant, number
9 (39) - -
(%)
Anti-depressant, number
6 (26) 25 (100) -
(%)

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Anti-psychotic 16 (69) - -
Benzodiazepine 1 (4) - -
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Supraliminal Task - Motion
Outliers, mean ± SD 6.87 ± 7.38 3.48 ± 3.98 3.84 ± 5.78 2.41 NS
Subliminal Task - Motion
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Outliers, mean ± SD 6.48 ± 8.36 5.56 ± 5.86 4.04 ± 4.21 0.920 NS

Abbreviations: BP, Bipolar Disorder; DASS, Depression Anxiety and Stress Scale; HDRS17, 17-
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YMRS, Young Mania Rating Scale.

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Table 2. Differences in amygdala activation for both supraliminal and subliminal emotional
face processing for euthymic Bipolar Disorder (BP) & remitted Major Depressive Disorder
(MDD) groups. Effects significant at cluster level FDR correction (p<0.05) are shown. (L-
left; n.s. – not significant)

MNI space
Region of increased Cluster
Emotion Direction X Y Z Z-score P-value
activation Size

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Supraliminal
All Emotions Main Effect (BP < MDD) L Amygdala -24 -2 -28 31 2.81 0.003
Threat Main Effect (BP < MDD) L Amygdala -24 0 -28 32 2.67 0.004

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Sad BP < MDD L Amygdala -24 0 -28 34 2.73 0.003
Happy BP vs MDD n.s. n.s. n.s. n.s. n.s. n.s. n.s.
Neutral BP < MDD L Amygdala -24 0 -28 50 3.02 0.001

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Subliminal
All Emotions Main Effect (BP < MDD) L Amygdala -22 -6 -18 193 3.74 <0.001
Threat Main Effect (BP < MDD) L Amygdala -22 -4 -18 197 4.08 <0.001
Sad BP < MDD L Amygdala -20 -4 -18 196 3.51 <0.001

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Happy BP < MDD L Amygdala -22 -4 -18 163 3.23 0.001
Neutral BP < MDD L Amygdala -22 -4 -18 160 3.13 0.001
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Table 3. Differences in left amygdala connectivity in the negative and positive affect
networks for both supraliminal and subliminal emotional face processing for euthymic
Bipolar Disorder (BP) & remitted Major Depressive Disorder (MDD) groups. Effects
significant at cluster level FDR correction (p<0.05) are shown. (L-left; R-right; mOFC-
medial orbitofrontal cortex; sgACC- subgenual anterior cingulate cortex; pgACC- pregenual
anterior cingulate cortex; n.s. – not significant)

MNI space
Cluster
Emotion Direction Region X Y Z Z-score P-value

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Size
Supraliminal
Negative Affect Network
Negative Affect Main Effect (BP > MDD) sgACC 4 26 -14 81 2.33 0.01

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Negative Affect Main Effect (BP > MDD) R Amygdala 28 0 -18 137 2.78 0.003
Negative Affect Main Effect (BP < MDD) R Hippocampus 30 -24 -8 174 2.26 0.012
Negative Affect Interaction L Insula -36 -2 4 158 2.27 0.012

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Threat Main Effect (BP < MDD) R Hippocampus 34 -24 -8 95 2.35 0.009
Threat Interaction L Insula -32 16 -18 75 2.41 0.008
Sad BP > MDD R Insula 44 22 0 369 2.63 0.004

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Sad BP < MDD L Hippocampus -30 -20 -14 150 2.68 0.004
Sad BP < MDD R Hippocampus 24 -16 -12 114 2.28 0.011
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Neutral BP < MDD R Amygdala 28 -2 -18 194 3.26 0.001
Neutral BP < MDD R Hippocampus 28 -6 -18 223 2.77 0.003
Positive Affect Network
Happy BP < MDD mOFC 0 56 -2 256 2.86 0.002
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Happy BP < MDD L Caudate -12 4 20 119 2.64 0.004

Happy BP < MDD L Putamen -26 -6 -8 110 2.66 0.005
Subliminal
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Negative Affect Network

Negative Affect Main Effect (BP < MDD) pgACC -2 48 4 241 2.72 0.003
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Negative Affect Main Effect (BP < MDD) sgACC 2 24 -8 90 1.95 0.026
Negative Affect Main Effect (BP < MDD) L Insula -38 20 2 302 2.15 0.016
Negative Affect Main Effect (BP < MDD) R Insula 40 10 6 77 2.2 0.014
Negative Affect Main Effect (BP < MDD) R Amygdala 26 0 -16 95 2.07 0.019
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Negative Affect Main Effect (BP < MDD) R Hippocampus 38 -30 -8 305 2.97 0.002
Negative Affect Interaction pgACC 0 48 8 138 2.53 0.006
Negative Affect Interaction L Insula -36 4 -6 879 3.44 <0.001
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Negative Affect Interaction R Insula 36 14 -6 872 3.92 <0.001

Negative Affect Interaction R Hippocampus 32 -20 -8 184 3.44 <0.001
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Threat Main Effect (BP < MDD) L Insula -38 18 2 690 2.99 0.001
Threat Main Effect (BP < MDD) R Insula 40 10 6 652 3.28 0.001
Threat Main Effect (BP < MDD) R Hippocampus 28 -20 -10 435 3.2 0.001
Threat Interaction L Insula -38 -4 -2 519 2.8 0.003
Threat Interaction R Insula 34 18 -6 244 2.93 0.002
Sad BP > MDD L Insula -30 10 -20 80 3.23 0.001
Sad BP < MDD R Amygdala 28 -2 -22 92 2.9 0.002
Neutral BP < MDD R Hippocampus 22 -36 4 129 2.44 0.007
Positive Affect Network
Happy BP < MDD mOFC 2 48 0 82 2.16 0.015
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Table 4. Classification models to classify BP & MDD patient groups: regression and cross
validation analyses. * indicates connectivity measures. (L-left; BP-Bipolar disorder; MDD-
Major Depressive Disorders; n.s. – not significant).

Overall Model Model Prediction Accuracy

Emotion Measure
Summary parameters (Cross-Validated Accuracy)
% % %
χ2 P β P
accuracy specificity sensitivity
Model 1: Supraliminal

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95.5 90.9 93.2
Full Model 42.89 0.000
(88.6) (90.9) (86.4)
Fear L Amygdala -2.59 0.037
Happy *mOFC -1.82 0.066

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Happy *L Caudate -5.46 0.023
Neutral *R Amygdala -7.17 0.015
Sad *R insula 3.43 0.041

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Model 2: Subliminal
81.8 81.8 81.8
Full Model 35.83 0.000
(79.5) (81.8) (77.2)
Anger L Amygdala -7.91 0.008

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Disgust *R Insula -1.96 0.038
Sad *L Insula -3.64 0.02
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Sad *R Amygdala 6.09 0.02
Model 3: Supraliminal + Subliminal
86.4 86.4 86.4
Full Model 34.02 0.000
(81.8) (77.2) (86.4)
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Happy *mOFC -1.19 0.049

Anger L Amygdala -6.64 0.008
Sad *L Insula -4.10 0.004
Sad *R Amygdala 4.68 0.022
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Korgaonkar et al. Supplement

Amygdala Activation and Connectivity to Emotional Processing Distinguishes

Asymptomatic Patients With Bipolar Disorder and Unipolar Depression

Supplementary Information

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Table of Contents

Supplementary Methods

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Supplementary Results

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Table S1. Posthoc comparisons in amygdala activation for the individual threat emotions.

Table S2. Posthoc comparisons in left amygdala connectivity in the negative affect network for
the individual threat emotions.

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Table S3. Whole brain comparisons for supraliminal emotion processing.
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Table S4. Whole brain comparisons for subliminal emotion processing.

Figure S1. Activation levels during supraliminal emotion face processing for euthymic bipolar
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disorder (BP), remitted major depressive disorder (MDD) and healthy control (HC) groups from
the whole brain analyses.
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Table S5. BP vs. HC comparisons in amygdala activation for emotion processing.

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Table S6. MDD vs. HC comparisons in amygdala activation for emotion processing.

Table S7. BP vs. HC comparisons in left amygdala connectivity for emotion processing.
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Table S8. MDD vs. HC comparisons in left amygdala connectivity for emotion processing.

Table S9. BP vs MDD comparisons for emotions relative to neutral.

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Table S10. Correlations of age of diagnosis/onset and number of depressive episodes with
activation and connectivity measures that differentiated the BP and MDD groups.
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Table S11. Impact of medication on neural measures differentiating the BP and MDD group.

Supplementary References

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Korgaonkar et al. Supplement

Supplementary Methods

fMRI pre-processing methodology

Data was pre-processed and analysed using SPM8 (http://www.fil.oin.ucl.ac.uk/spm) statistical

parametric mapping software. During pre-processing, data was corrected for motion by

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realigning and unwarping the images to the first image of the task run. A mean image was

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generated for the fMRI time series and normalised to the T1-weighted structural scan, using the

FMRIB linear registration tool (FLIRT). Subsequently, the FMRIB non-linear registration tool

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(FNIRT) was used to normalise the T1-weighted data to standard space. The normalisation warps

from these steps were used subsequently in functional to standard space transformations. To

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account for any physiological noise, average signal using a mask from the CSF and white matter
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was removed from the motion-corrected fMRI time series. This data was then smoothed using an

8mm Gaussian kernel and a high-pass filter using a cut-off period of 128 seconds. A
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hemodynamic response convolved box car function was used to model the blood oxygen level
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dependent (BOLD) responses for each emotion block. Before analyses, we screened for gross
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motion artifacts in our dataset using the Artifact Detection Tools (ART,

www.nitrc.org/projects/artifact_detect/). The motion parameters (three translation and three

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rotational) estimated during the realignment pre-processing stage were used to identify any

problematic fMRI volumes in each participant’s entire scan. A volume frame was defined as an
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outlier (artifact) if the head displacement in x, y or z direction was greater than .5mm from the
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previous frame, or if the global mean intensity in the volume was greater than 3 standard

deviations from the mean image intensity for the entire scan. Participant data (n=3) with more

than 25% outlier volume were rejected from further analysis. Participant movement was also

evaluated for any significant differences across groups (Table 1).

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Korgaonkar et al. Supplement

fMRI connectivity analysis using generalized psychophysiological interactions (gPPI)

A generalized context-dependent PPI (gPPI) model identifies how task-specific changes in the

BOLD signal across difference regions in the brain interact over time (1). This model generates a

regressor of each task condition’s onset times, in this case for each emotion, and individually

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convolves this with the hemodynamic response function (HRF) to form a psychological

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interaction term. This term is then multiplied by the estimated neural activity of the seed region

(the physiological regressor), derived from the deconvolved BOLD signal of this seed (2), and

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other relevant covariates i.e. motion regressors. This differs from a standard PPI where the

psychological regressor is created by producing the product of the condition onset times and a

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weighting/contrast vector before it is multiplied with the physiological term (3). The gPPI
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approach therefore generates a more accurate model of the interaction between multiple

conditions and neural activity across the entire experimental space (1). This model allows us to
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explore the correlation between the BOLD response in various voxels or ROIs through the brain
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and neural activity in the seed region during any of multiple task conditions, providing a measure
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of task-related connectivity. In our analysis, the left amygdala was chosen as the seed and

connectivity maps corresponding to the interaction term of the emotion with this seed time-series
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was estimated for every participant. Second level analyses were then performed to evaluate

group comparisons using these connectivity maps as described in the main text.
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Korgaonkar et al. Supplement

Supplementary Results

Comparisons in left amygdala activation and connectivity for individual threat emotions

Following a significant main effect of group in an ANOVA across the threat facial emotions, we

ran post hoc voxel-wise group comparisons for activation and connectivity related to the

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amygdala for each of the individual threat emotions i.e. anger, disgust and fear. These results are

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summarized in Table S1 & S2 below. BP group had a lower left amygdala activation and lower

subliminal amygdala-insula & amygdala-hippocampal connectivity relative to MDD.

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Table S1. Posthoc comparisons in amygdala activation for the individual threat emotions.

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Effects significant at cluster level FDR correction are shown. (L-left; BP-Bipolar Disorder;
MDD – Major Depressive Disorder; n.s. – not significant)
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Cluster
Emotion Direction Region X Y Z Z-score P-value
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Size
Supraliminal
Anger n.s. n.s. n.s. n.s. n.s. n.s. n.s n.s
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Disgust BP < MDD L Amygdala -24 0 -28 29 2.75 0.003

Fear BP < MDD L Amygdala -24 0 -28 43 2.9 0.002
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Subliminal
Anger BP < MDD L Amygdala -22 -4 -18 188 3.23 0.001
Disgust BP < MDD L Amygdala -26 -6 -16 184 3.34 <0.001
Fear BP < MDD L Amygdala -24 -6 -16 181 3.43 <0.001
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Table S2. Posthoc comparisons in left amygdala connectivity in the negative affect network for
the individual threat emotions. Effects significant at cluster level FDR correction are shown. (L-
left; R-right; BP-Bipolar Disorder; MDD – Major Depressive Disorder; n.s. – not significant)

MNI space
Cluster
Emotion Direction Region X Y Z Z-score P-value

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Size
Supraliminal
Anger n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
Disgust n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.

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Fear n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
Subliminal

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Anger BP < MDD L Insula -40 0 6 981 3.01 0.001
Anger BP < MDD R Insula 44 18 -8 673 3.13 0.001
Anger BP < MDD R Hippocampus 34 -20 -8 173 2.88 0.002
Disgust BP < MDD L Insula -38 16 2 354 2.79 0.003

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Disgust BP < MDD R Insula 38 22 4 628 3.21 0.001
Disgust BP < MDD R Hippocampus 38 -22 -8 284 2.61 0.005
Fear
Fear
BP < MDD
n.s.
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R Hippocampus
n.s.
30
n.s.
-22
n.s.
-12
n.s.
329
n.s.
3.11
n.s.
0.001
n.s.
Fear n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
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Exploratory whole brain voxel-wise comparisons for euthymic bipolar disorder (BP) vs.

remitted major depressive disorder (MDD)

We performed a voxel-wise ANOVA across all emotions to evaluate any other significant effects

(main effect of group and group-by-emotion interactions) beyond the amygdala for both

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supraliminal and subliminal emotion processing. As done for the ROI analysis, a cluster level

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FDR correction was employed using a whole brain grey matter mask. A threshold of p<0.001 &

19 voxel clusters was estimated using Monte Carlo simulations to determine a significant alpha

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at 0.05 using this mask. These results are summarized in Table S3 & S4 below. Figure S1 shows

the group-wise activation levels for the individual emotions for a selected significant cluster

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from the analysis.
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Table S3. Whole brain comparisons for supraliminal emotion processing. Effects significant at
cluster level FDR correction are shown. (L-left; R-right; BP-Bipolar Disorder; MDD – Major
Depressive Disorder; n.s. – not significant)
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MNI space
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Cluster
Emotion Direction Region X Y Z Z-score P-value
Size
All Emotions Main Effect (BP > MDD) Occipital_Inf_R 32 -86 -4 522 5.01 <0.001
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SupraMarginal_R 62 -28 50 127 4.45 <0.001

Precentral_L -26 -14 78 100 4.08 <0.001
Temporal_Inf_R 58 -4 -38 156 4.06 <0.001
Frontal_Sup_Medial_R 10 50 28 222 4.05 <0.001
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Calcarine_R 6 -90 4 67 4.03 <0.001

Lingual_L -22 -64 -8 368 4.02 <0.001
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Frontal_Sup_R 16 26 44 128 3.85 <0.001

Fusiform_R 26 -54 -14 160 3.68 <0.001
Temporal_Inf_L -52 -6 -42 54 3.65 <0.001
Frontal_Inf_Oper_R 38 14 36 32 3.48 <0.001
Lingual_R 6 -76 -4 57 3.27 0.001
All Emotions Main Effect (BP < MDD) Cerebelum_9_R 16 -48 -44 140 4.1 <0.001
Cerebelum_Crus2_L -36 -70 -42 35 3.84 <0.001
All Emotions Interaction n.s. n.s. n.s. n.s. n.s. n.s. n.s.

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Table S4. Whole brain comparisons for subliminal emotion processing. Effects significant at
cluster level FDR correction are shown. (L-left; R-right; BP-Bipolar Disorder; MDD – Major
Depressive Disorder; n.s. – not significant)

MNI space
Cluster P-value
Emotion Direction Region of activation X Y Z Z-score

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Size (uncorrected)
All Emotions Main Effect (BP > MDD) Occipital_Mid_L -16 -88 -4 139 3.52 <0.001
Calcarine_R 20 -92 4 22 3.3 <0.001
Calcarine_L 4 -80 12 30 3.28 0.001

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All Emotions Main Effect (BP < MDD) Cerebelum_Crus1_R 22 -76 -36 62 4.07 <0.001
Frontal_Inf_Orb_R 60 24 -8 25 3.89 <0.001
Amygdala_L -22 -6 -18 162 3.74 <0.001

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ParaHippocampal_L -14 -30 -12 39 3.69 <0.001
Frontal_Mid_L -32 18 32 56 3.6 <0.001
Supp_Motor_Area_R 4 4 62 23 3.45 <0.001

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Frontal_Inf_Tri_L -42 24 -2 34 3.36 <0.001
Cerebelum_Crus1_R 48 -60 -38 27 3.29 <0.001
All Emotions Interaction
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Angular_R 50 -72 42 38 3.69 <0.001
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Figure S1. Activation levels during supraliminal emotion face processing for euthymic bipolar
disorder (BP), remitted major depressive disorder (MDD) and healthy control (HC) groups from
the whole brain analyses. Brain image shows significant differences in activation for the main
effect of emotion comparing the BP & MDD groups (cluster level FDR corrected p<0.05).
* indicates significant group differences.

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Comparisons of each patient group relative to healthy controls (HC)

We performed voxel-wise comparisons for activations and connectivity related to the amygdala

for both euthymic bipolar disorder (BP) and remitted major depressive disorder (MDD) groups

relative to controls in separate analyses. As done in the main text, a cluster level FDR correction

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was employed. As there were significant differences in DASS depression scores for both BP and

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MDD relative to controls, we extracted beta values for the significant clusters and evaluated if

these neural differences survived after controlling for the DASS measures.

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Table S5. BP vs. HC comparisons in amygdala activation for emotion processing. Effects

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significant at cluster level FDR correction are shown. None of the group differences remained
significant after controlling for DASS measures. (L-left; BP-Bipolar Disorder; HC – Healthy
controls; n.s. – not significant). AN
MNI space
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Cluster
Emotion Direction Region X Y Z Z-score P-value
Size
Supraliminal
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Threat BP vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s

Sad BP vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s
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Happy BP vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s

Neutral BP vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s
Anger BP vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s
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Disgust BP vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s

Fear BP vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s
Subliminal
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Threat Main Effect (BP < HC) L Amygdala -30 -4 -16 154 2.28 0.011
Sad BP < HC L Amygdala -20 2 -20 173 2.67 0.004
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Happy BP < HC L Amygdala -20 2 -20 43 2.24 0.013

Neutral BP < HC L Amygdala -22 2 -18 130 2.39 0.008
Anger BP < HC L Amygdala -20 2 -20 78 2.06 0.02
Disgust BP < HC L Amygdala -28 -6 -18 93 2.24 0.013
Fear BP < HC L Amygdala -28 -6 -18 148 2.3 0.011

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Table S6. MDD vs. HC comparisons in amygdala activation for emotion processing. Effects
significant at cluster level FDR correction are shown. None of the group differences remained
significant after controlling for DASS measures. (L-left; MDD – Major Depressive Disorder; HC
– Healthy controls; n.s. – not significant)

MNI space

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Cluster P-value
Emotion Direction Region of activation X Y Z Z-score
Size (uncorrected)
Supraliminal

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Threat MDD vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s
Sad MDD vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s
Happy MDD vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s

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Neutral MDD vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s
Anger MDD vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s
Disgust MDD vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s

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Fear MDD vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s
Subliminal
Threat
Sad
Main Effect (MDD > HC)
MDD vs HC
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L Amygdala
n.s.
-22
n.s.
-8
n.s.
-14
n.s.
61
n.s.
2.71
n.s
0.003
n.s
Happy MDD vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s
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Neutral MDD vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s
Anger MDD > HC L Amygdala -20 -8 -16 38 2.17 0.015
Disgust MDD > HC L Amygdala -22 -8 -14 49 2.26 0.012
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Fear MDD > HC L Amygdala -20 -6 -14 27 2.08 0.019

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Table S7. BP vs. HC comparisons in left amygdala connectivity for emotion processing.
Effects significant at cluster level FDR correction are shown. * indicates group differences that
remained significant after controlling for DASS measures. (L-left; R-Right; BP-Bipolar
Disorder; HC-Healthy controls; n.s. – not significant).

MNI space

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Cluster P-value
Emotion Direction Region of activation X Y Z Z-score
Size (uncorrected)
Supraliminal

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Negative Affect Network
Threat BP vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s
Sad BP < HC L Hippocampus -30 -18 -22 192 3.27 0.001

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*Neutral BP < HC R Amygdala 24 -8 -12 184 2.97 0.001
Neutral BP < HC R Hippocampus 32 -20 -8 812 2.94 0.002
Positive Affect Network

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*Happy BP < HC L Putamen -30 0 -8 109 3.26 0.001
Subliminal
Negative Affect Network
Threat Main Effect (BP < HC)
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L Insula -38 12 14 384 2.95 0.002
Threat Main Effect (BP < HC) R Insula 28 22 -8 210 3.31 <0.001
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*Threat Main Effect (BP < HC) R Hippocampus 24 -18 -12 460 3.19 0.001
Threat Interaction L Insula -38 -4 2 75 2.18 0.015
Sad BP vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s
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Neutral BP vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s

Posthoc Comparisons for Threat
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Anger BP < HC L Insula -42 0 8 292 3.06 0.001

*Anger BP < HC R Insula 30 18 -10 516 2.88 0.002
Anger BP < HC R Hippocampus 24 -18 -12 143 2.15 0.016
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Disgust BP vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s

*Fear BP < HC L Insula -26 22 4 354 3.45 0
*Fear BP < HC R Insula 28 22 -8 178 2.58 0.005
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*Fear BP < HC R Hippocampus 28 -24 -8 433 2.6 0.005

Positive Affect Network
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Happy BP vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s

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Table S8. MDD vs. HC comparisons in left amygdala connectivity for emotion processing.
Effects significant at cluster level FDR correction are shown. * indicates group differences that
remained significant after controlling for DASS measures. (L-left; MDD-Major Depressive
Disorder; HC-Healthy controls; n.s. – not significant).

MNI space

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Cluster P-value
Emotion Direction Region of activation X Y Z Z-score
Size (uncorrected)
Supraliminal

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Negative Affect Network
Threat Interaction L Insula -30 -28 22 108 3.01 0.001
Threat Interaction R Hippocampus 18 -12 -16 82 3.15 0.001

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Sad MDD > HC R Hippocampus 30 -36 -4 98 2.19 0.014
Neutral MDD vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s
Posthoc Comparisons for Threat

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Anger MDD vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s
Disgust MDD vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s
Fear MDD vs HC
Positive Affect Network
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n.s. n.s. n.s. n.s. n.s. n.s n.s

*Happy MDD > HC mOFC 8 48 -12 178 2.68 0.004

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Subliminal
Negative Affect Network
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Threat Interaction R Hippocampus 24 -14 -22 76 2.69 0.004

Sad MDD vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s
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Neutral MDD vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s

Posthoc Comparisons for Threat
Anger MDD vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s
Disgust MDD vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s
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Fear MDD vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s

Positive Affect Network
Happy MDD vs HC n.s. n.s. n.s. n.s. n.s. n.s n.s
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Voxel-wise comparisons in activations for each emotion relative to neutral

In our main analysis, we used contrasts of each individual emotion relative to the baseline

average. In order to compare with previous fMRI studies of emotion processing using neutral as

baseline, we evaluate here BP vs. MDD group differences using each individual emotion relative

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to neutral for both supraliminal and subliminal emotion tasks. Voxel-wise group comparisons

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were performed using a cluster level FDR correction in the amygdala. These results are

summarized in Table S9 below. The only significant differences (after FDR correction) were for

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supraliminal processing of happy (BP>MDD) & subliminal processing of sad (BP<MDD)

emotions. We note that the direction of findings for happy using the neutral contrast (BP>MDD)

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were different from those using the baseline average (BP<MDD). This could be related due to a
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significant difference in BP and MDD observed for neutral face processing (table 2 in main

manuscript).
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Table S9. BP vs MDD comparisons for emotions relative to neutral. * indicates group
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differences that remained significant at cluster level FDR correction. (L-left; BP-Bipolar
disorder; MDD-Major Depressive Disorder; n.s. – not significant).
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MNI space
Cluster
Emotion Direction Region X Y Z Z-score P-value
Size
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Supraliminal
Threat Main Effect (BP > MDD) L Amygdala -24 4 -18 2 1.73 0.042
Sad BP > MDD L Amygdala -30 -4 -16 3 1.69 0.045
*Happy BP > MDD L Amygdala -28 -4 -18 116 3.12 0.001
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Anger BP > MDD L Amygdala -24 2 -22 22 1.9 0.029

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Disgust BP vs MDD L Amygdala n.s. n.s. n.s. n.s. n.s n.s

Fear BP vs MDD L Amygdala n.s. n.s. n.s. n.s. n.s n.s
Subliminal
Threat Main Effect (BP < MDD) L Amygdala -30 -4 -22 10 1.76 0.039
*Sad BP < MDD L Amygdala -16 -6 -18 45 2.3 0.011
Happy BP vs MDD L Amygdala n.s. n.s. n.s. n.s. n.s n.s
Anger BP < MDD L Amygdala -30 0 -28 15 1.95 0.026
Disgust BP > MDD L Amygdala -30 -4 -20 22 1.97 0.024
Fear BP vs MDD L Amygdala n.s. n.s. n.s. n.s. n.s n.s

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Evaluation of neural measures as trait markers

To further test if the identified neural measures were trait markers for either of the disorders, we

investigated whether there were any correlations for the significant neural measures (activation

and connectivity measures that were different between the groups) with age of diagnosis (for BP

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patients) or age of onset (for MDD patients), and number of depressive episodes which represent

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clinical severity of the disorders. We hypothesized that trait neural measures would become more

severe with clinical severity. Only amygdala activation for disgust, fear, sad and neutral

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emotions from the supraliminal task were significantly negatively correlated with age of

diagnosis and number of episodes in the BP group i.e. lower activations for patients with more

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number of episodes or a later age of diagnosis. In comparison, for the MDD group, no significant
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associations with activations were observed. However, bilateral amygdala connectivity to

supraliminal viewing of neutral and left amygdala-right insula connectivity to subliminal

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viewing of disgust was negatively correlated with number of episodes (Table S10).
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Table S10. Correlations of age of diagnosis/onset and number of depressive episodes with
activation and connectivity measures that differentiated the BP and MDD groups. * indicates
connectivity measures. (L-left; BP-Bipolar disorder; MDD-Major Depressive Disorder; n.s. – not
significant).

Level of
Emotion Region Pearson Correlation P

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Processing
Bipolar Patients
Age of Diagnosis
Supraliminal Disgust L Amygdala -0.529 0.035

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Supraliminal Neutral L Amygdala -0.510 0.044
Supraliminal Sad L Amygdala -0.530 0.035
Number of Episodes

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Supraliminal Disgust L Amygdala -0.580 0.012
Supraliminal Fear L Amygdala -0.470 0.049
Supraliminal Neutral L Amygdala -0.586 0.011
Supraliminal Sad L Amygdala -0.546 0.019

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MDD Patients
Age of Onset

Number of Episodes
n.s. AN n.s. n.s. n.s. n.s.

Supraliminal Neutral *R Amygdala -0.452 0.035

Subliminal Disgust *R Insula -0.436 0.042
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Evaluation of the effect of medication use

Evaluation effect of medications between the groups is not possible as the two patient groups

were on different types of medications, so we evaluate effect of medication within each group

separately. In deciding how to appropriately quantify medication, a series of approaches were

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considered. Firstly we considered an analysis of lithium medication levels, but did not have

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blood serum levels to accurately account for medication load. We then considered splitting our

bipolar group (based on those who were and those who were not taking mood stabilizers) to

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investigate the effects of the mood stabilizer, but did not have enough patients not on lithium

(n=5). Finally we decided the best, and most evidence based, approach would be analyzing anti-

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psychotic medication, standardized through dose equivalents of chlorpromazine, as done in (4),
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because we had holistically the most information for our BP group with this medication type. As

our patient groups did not share a medication type, our MDD group required another analysis
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approach. We considered a dosage load analysis but ultimately decided against it as there was
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types. We decided on pooling patients taking SSRIs (n = 10) and comparing their brain measures

against those taking an SNRI class of antidepressant (n = 12) in a two-sample t-test. There were
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no differences between these groups in age or HDRS scores. Results are summarized in Table

S11. Medication was found to impact only one connectivity measure (amygdala-left insula for
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subliminal disgust stimuli) in the BP group whereas two neural measures (amygdala activation
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for supraliminal sad and amygdala-left insula connectivity for supraliminal angry faces) were

different between MDDs on SSRI vs. SNRI medications.

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Table S11. Impact of medication on neural measures differentiating the BP and MDD group. *
indicates connectivity measures. (L-left; BP-Bipolar disorder; MDD-Major Depressive Disorder;
n/a – not applicable).
Level of Pearson
Emotion Region P t
Processing Correlation

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Bipolar Patients

Anti-Psychotic dosage

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Subliminal Disgust *L Insula 0.584 0.014 n/a

MDD Patients

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SSSR v SNRI

Supraliminal Sad L Amygdala n/a 0.019 2.629

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Supraliminal Anger * L Insula n/a 0.012 -2.817

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Supplementary References

1. McLaren DG, Ries ML, Xu G, & Johnson SC (2012): A generalized form of context-
dependent psychophysiological interactions (gPPI): a comparison to standard approaches.
NeuroImage 61(4):1277-1286.
2. Gitelman DR, Penny WD, Ashburner J, & Friston KJ (2003): Modeling regional and

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psychophysiologic interactions in fMRI: the importance of hemodynamic deconvolution.
NeuroImage 19(1):200-207.
3. Friston KJ, Buechel C, Fink GR, Morris J, Rolls E, Dolan RJ (1997):
Psychophysiological and modulatory interactions in neuroimaging. NeuroImage 6:218-

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29.
4. Leucht S, Samara M, Heres S, Patel MX, Woods SW, & Davis JM (2014): Dose
Equivalents for Second-Generation Antipsychotics: The Minimum Effective Dose

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Method. Schizophrenia Bulletin 40(2):314-326.

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