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The Journal of , Vol 15, No 8 (August), 2014: pp 815-826 Available online at www.jpain.org and www.sciencedirect.com

Original Reports

Altered Resting State Connectivity of the in Individuals With

Eric Ichesco,* Tobias Schmidt-Wilcke,*,** Rupal Bhavsar,*,y Daniel J. Clauw,* Scott J. Peltier,z Jieun Kim,x Vitaly Napadow,x,{,k Johnson P. Hampson,* Anson E. Kairys,*,yy David A. Williams,* and Richard E. Harris* *Department of Anesthesiology, Chronic Pain and Fatigue Research Center, University of Michigan, Ann Arbor, Michigan. yNeurology Department, University of Pennsylvania, Philadelphia, Pennsylvania. z Functional MRI Laboratory, University of Michigan, Ann Arbor, Michigan. xMGH/MIT/HMS Athinoula A. Martinos Center for Biomedical Imaging, Charlestown, Massachusetts. {Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts. kDepartment of Radiology, Logan College of Chiropractic, Chesterfield, Missouri. **Department of Neurology, Bergmannsheil, Ruhr University Bochum, Bochum, Germany. yyDepartment of Psychology, University of Colorado Denver, Denver, Colorado.

Abstract: The insular cortex (IC) and (CC) are critically involved in pain . Previously we demonstrated that fibromyalgia (FM) patients have greater connectivity between the insula and at rest, and that changes in the degree of this connectivity were associated with changes in the intensity of ongoing clinical pain. In this study we more thoroughly evaluated the degree of resting-state connectivity to multiple regions of the IC in individuals with FM and healthy controls. We also investigated the relationship between connectivity, experimental pain, and current clinical chronic pain. Functional connectivity was assessed using resting-state functional magnetic resonance imaging in 18 FM patients and 18 age- and sex-matched healthy controls using predefined seed regions in the anterior, middle, and posterior IC. FM patients exhibited greater con- nectivity between 1) right mid IC and right mid/posterior CC and right mid IC, 2) right posterior IC and left CC, and 3) right anterior IC and left superior temporal . Healthy controls displayed greater connectivity between left anterior IC and bilateral /anterior cingulate cortex; and left posterior IC and right . Within the FM group, greater connectivity between the IC and CC was associated with decreased pressure-pain thresholds. Perspective: These data provide further support for altered resting-state connectivity between the IC and other regions known to participate in pain perception/modulation, which may play a patho- genic role in conditions such as FM. We speculate that altered IC connectivity is associated with the experience of chronic pain in individuals with FM. ª 2014 by the American Pain Society Key words: Fibromyalgia, chronic pain, resting-state connectivity, insular cortex, cingulate cortex.

Received February 17, 2014; Revised April 7, 2014; Accepted April 15, All other authors have no conflicts of interest to disclose. 2014. Supplementary data accompanying this article are available online at E.I. and T.S.-W. contributed equally to this study. www.jpain.org and www.sciencedirect.com. This study was funded in part by National Institutes of Health/National Address reprint requests to Eric Ichesco, BS, Department of Anesthesi- Center for Complementary and Alternative Medicine grant K01-AT- ology, Chronic Pain and Fatigue Research Center, University of Michigan, 01111-01 and in part by a grant from Pfizer Incorporated, Groton, CT Ann Arbor, MI 48105. E-mail: [email protected] (no. A0081211). T.S.-W. was supported by a grant of the Deutsche For- 1526-5900/$36.00 schungsgemeinschaft (GZ: SchM 2665/1-1). R.E.H. is supported by grants ª from the Dana Foundation and the Department of Defense (Army grant: 2014 by the American Pain Society DAMD-W81XWH-07-2-0050), and has previously consulted for Pfizer Inc. http://dx.doi.org/10.1016/j.jpain.2014.04.007 D.J.C. has previously consulted for Pfizer Inc. V.N. was supported by Na- tional Center for Complementary and Alternative Medicine, National In- stitutes of Health (R01-AT004714, P01-AT002048).

815 816 The Journal of Pain Functional Connectivity in Fibromyalgia he insular cortex (IC) and cingulate cortex (CC) play Here we investigate whether these IC-CC connectivity pivotal roles in integrating multimodal informa- patterns are also seen in FM patients and whether these tion involved in sensorimotor, emotional, and ho- relationships are related to the hyperalgesia/allodynia T 9,11,46 meostatic functions. Functional brain imaging these patients experience. We used an approach similar studies in often show a coactivation of IC and to that of Taylor et al, looking specifically at seed-based CC in a variety of tasks, including attention, decision IC-CC and IC-IC connectivity in a sample of FM patients making, -recognition, and time perception.10,13,26 compared to HCs. This analysis expands on our previ- Both regions are critically involved in pain perception ously published connectivity results by applying the Tay- as evidenced by brain imaging studies.36 lor et al seeds30,31 to FM and linking connectivity to Fibromyalgia (FM) is a chronic pain state characterized by evoked pain data. Given previous findings, we widespread nonarticular pain, stiffness, sleep and mood hypothesized that differences in IC-CC and IC-IC connec- disturbances, and fatigue.50 Impaired antinociception tivity would be detected in FM and that this might pro- and augmented central processing of nociceptive input vide further insights into the central neural correlates of have been considered to be partially responsible for pain chronic pain. in FM.48 Support for FM’s being a predominantly centrally Results from this manuscript were previously pre- mediated pain state comes from quantitative sensory sented at the 2012 Annual Scientific Meeting of the testing and studies using experi- American Pain Society.23 mentally induced pain stimuli, indicating both diffuse hy- peralgesia and allodynia.8,17 Despite their heuristic value, these earlier studies examined primarily experimentally Methods induced pain rather than ongoing clinical pain. With the emergence of advanced neuroimaging Subjects methods, new approaches to neurobiological correlates This study was approved by the medical institution of ongoing clinical pain have been developed. One review board of the University of Michigan, and all sub- such method is functional connectivity magnetic reso- jects read and signed an informed consent form prior to nance imaging (fcMRI), a noninvasive technique applied participation. Using the fcMRI protocol described in in awake humans either at rest (ie, resting-state connec- detail (see below), we investigated 18 female FM tivity) or during task. With resting-state analyses, low- patients (age, 35.8 6 12.0 years) and 18 age- and frequency (<.1 Hz) temporal correlations in the MRI sex-matched HCs (32.3 6 11.3 years). A subset of these signal are assessed across various brain regions. These FM patients and HCs had been part of a previously re- low-frequency fluctuations are thought to be function- ported study using different methodologies to investi- ally relevant indices of connectivity between brain re- gate hypotheses different from those included in the gions subserving similar or related brain functions.2 present study.30,31 Inclusion criteria for FM patients Currently, fcMRI has been investigated in few chronic were as follows: 1) met the 1990 American College of pain states.6 When investigating fcMRI in patients with Rheumatology criteria for FM,50 2) disease duration of temporomandibular disorders, we found greater resting- at least 1 year and continued presence of pain for more state fcMRI between the anterior IC and the pregenual than 50% of each day, 3) age between 18 and 75 years, anterior cingulate cortex (ACC) in patients when compared 4) right-handedness, and 5) capable of giving informed to healthy controls (HCs).22 Our recent studies in FM have written consent. Participants were excluded from analysis suggested that resting connectivity between the IC and if any new treatments were introduced between consent- default mode network (DMN), a constellation of brain re- ing and imaging time points. Exclusion criteria for FM pa- gions activated during self-referential thinking, was corre- tients included 1) current use or history of taking opioid lated to current clinical pain at the time of the scan.30,31 or narcotic analgesics; 2) history of substance abuse; 3) Additionally, we reported in 2 longitudinal trials that concurrent autoimmune or inflammatory disease that decreases in IC to DMN connectivity in FM patients were caused pain, such as rheumatoid arthritis, systemic lupus associated with reductions in clinical pain.20,30 Although erythematosus, or inflammatory bowel disease; 4) con- whole brain searches were performed, our findings were current participation in other therapeutic trials; 5) preg- largely restricted to connectivity between the DMN and nant or currently a nursing mother; and 6) history of the IC in FM patients. In support of our model, greater psychiatric illness (eg, current , major DMN-IC connectivity, as well as strong association between depression with suicidal ideation, or substance abuse this connectivity and clinical pain, was also noted in chronic within the past 2 years) or current major depression. low back pain patients.27 HC subjects were age- and sex-matched to the FM pa- Taylor and colleagues recently investigated IC connec- tients. Inclusion criteria for HCs were 1) age between tivity in a sample of HCs to systematically explore IC con- 18 and 75, 2) right-handedness, 3) capable of giving writ- nectivity to other brain regions.43 Predefined seed ten informed consent, and 4) willingness to complete all regions were placed in the IC, and functional connectiv- study procedures. Exclusion criteria for the HC subjects ity analyses showed that 1) the anterior IC was connected were as follows: 1) met the 1990 American College of to both posterior ACC and anterior midcingulate cortex Rheumatology criteria for FM, 2) any chronic medical (MCC) regions, and 2) the mid/posterior IC was con- illness, including a psychiatric disorder (eg, psychosis, nected to the posterior MCC and posterior CC. schizophrenia, or delusional disorder), 3) a chronic pain Ichesco et al The Journal of Pain 817 disorder, and 4) current pregnancy. All FM patients and Descriptive words are arranged next to the HCs had no contraindications for participating in an numbers corresponding with varying levels of pain.15 fMRI scan. To elicit the GBS ratings, discrete pressure stimuli were applied to the subject’s right thumbnail using a hydraulic 2 Behavioral Data system connected to a hard rubber 1-cm circular probe.16,19 The following clinical features were assessed: demo- graphics (ie, age, duration of FM, and lifetime illness burden), clinical pain (ie, current pain at the time of Data Acquisition study visit), experimental pain (random noxious thumb- Resting state functional MRI (fMRI) data were acquired nail pressure stimuli), and allied mood and illness attribu- using a custom T2*-weighted spiral-in sequence [repeti- tions (ie, depression and ). tion time = 2.0, echo time = 30 ms, flip angle = 90 , matrix size = 64 64 with 43 slices, field of view = 20 cm, and Demographics 3.13 3.13 3 mm voxels], using a General Electric A standardized demographics form was used to record (GE) 3.0-T Signa scanner 9.0, VH3 with quadrature bird- age and sex (ie, all female in this study) as well as medical cage transmit-receive radio frequency coil (General Elec- status (eg, duration of FM in years). Lifetime illness tric, Milwaukee, WI). During the 6-minute resting-state burden was assessed using the Complex Medical Symp- fMRI acquisition period (180 scans), the subjects were tom Inventory,49 a 48-symptom checklist of comorbid asked to remain awake with eyes open. A fixation cross symptoms common to FM. was presented on the screen. Subjects were told to lie still and fixate on the cross throughout the scan. Minimal Clinical Pain cognitive tasks such as staring at a cross typically do not 18 The clinical pain experience of patients with FM was disrupt resting-state networks. Physiologic data were assessed using a visual analog scale (VAS) and the Pain collected simultaneously with fMRI data because cardio- Rating Index from the Short-Form McGill Pain Question- respiratory fluctuations are known to influence fMRI 3,7 naire (SF-MPQ).29 When completing the SF-MPQ, partic- intrinsic connectivity within several brain networks. ipants were asked to answer questions within each Respiratory volume data were collected by securing a section pertaining to their current pain. The VAS consists GE magnetic resonance–compatible chest plethysmo- of a 10-cm line anchored on the left with ‘‘no pain’’ and graph around each subject’s abdomen. Cardiac data on the right with ‘‘worst possible pain.’’ Participants in were collected using an infrared pulse oximeter (GE) the study were asked to rate their present FM pain by attached to the subject’s right middle finger. Partici- placing a tick along this line. The Pain Rating Index pants’ motion was minimized using foam pads placed component of the SF-MPQ consisted of 15 word descrip- around the head along with a forehead strap. In addi- tors (11 sensory and 4 affective). Participants rated these tion, high-resolution structural images were acquired [repetition time = 10.5 ms, echo time = 3.4 ms, inversion descriptors as either ‘‘none,’’ ‘‘mild,’’ ‘‘moderate,’’ or ‘‘se- vere,’’ giving a score of 0, 1, 2, or 3, respectively, for each time = 200 ms, flip angle = 25 , 24 cm field of view, matrix descriptor. The measures were added to yield sensory, af- size = 256 256, .94 .94 1.5 mm voxels, yielding 106 fective, and total scores. slices] using a spoiled gradient echo inversion recovery sequence. Inspection of individual T1 MR images re- Mood and Illness Attributions vealed no gross morphologic abnormalities for any patient or subject. Depression in the FM cohort was assessed with either the Hospital Anxiety and Depression Scale (n = 7) or the Center for Epidemiologic Studies Depression Scale (CES- Data Analysis D) (n = 11).39,51 FM patients were categorized to a low Data were preprocessed and analyzed using FSL (www. versus high level of depression by defining a high level fmrib.ox.ac.uk/fsl) and SPM (Statistical Parametric Map- as $8 symptoms on the Hospital Anxiety and ping) software packages, version 5 (Functional Imaging Depression Scale and $16 on the CES-D. State Anxiety Laboratories, London, UK), as well as the functional con- was assessed using the State-Trait Personality Inventory nectivity toolbox Conn (Cognitive and Affective Neuro- (Form Y),42 a refinement of the State-Trait Anxiety Inven- science Laboratory, Massachusetts Institute of tory. Technology, Cambridge, MA) running on Matlab 7.5 b (Mathworks, Sherborn, MA).47 On collection of the func- Experimental Pain Assessment tional data, cardiorespiratory artifacts were corrected for Prior to scanning, pressure-pain values eliciting ‘‘faint’’ using the RETROICOR21,37 algorithm in FSL. pain (.5 on the Gracely Box Scale [GBS]; see below and Preprocessing steps included motion correction Supplementary Fig 1), ‘‘mild’’ pain (7.5 on the GBS), and (realignment to the first image of the time series), ‘‘slightly intense’’ pain (13.5 on the GBS) were deter- normalization to the Montreal Neurological Institute mined for every subject using the multiple random stair- average brain included in the SPM software case (MRS) method as described previously.19,34 The GBS (generating 2 2 2 mm resolution images), and is a numerical scale that is used to evaluate present pain smoothing (convolution with a 6-mm full width at half intensity. This scale is composed of 21 boxes, sequentially maximum Gaussian kernel). Subject head motion was as- numbered beginning with 0 and ending with 20. sessed by evaluating 3 translations and 3 rotations for 818 The Journal of Pain Functional Connectivity in Fibromyalgia each scan. Translational thresholds were set to 62 mm, tivity maps, results were thresholded at an uncorrected whereas rotational thresholds were limited to 61.A voxel-level threshold of P < .00001, and deemed signifi- subject was to be excluded from the analysis if head mo- cant based on a corrected value of P < .05 on the cluster tion exceeded either of the thresholds in 1 of the 6 di- level. mensions. Based on the approach by Taylor et al,43 6 seed regions Analysis 2 were defined within the anterior, middle, and posterior We were then interested whether there were differ- IC bilaterally; seed regions were created as spheres (6- ences in connectivity between groups. To this end, 2 sam- mm-diameter) using MarsBaR software (http://marsbar. ple t-tests (controlling for age) for each seed region were sourceforget.net). For details on center coordinates performed. Because we were specifically looking for dif- and volumes, see Fig 1. Seed regions’ time series were ex- ferences between groups within the pain system and in tracted; white matter and cerebrospinal fluid signal, as brain areas involved in pain modulation, we allowed well as realignment parameters, were entered into the for these regions a less stringent threshold; differences analysis as covariates of no interest. A band-pass filter were deemed significant on the cluster level corrected (frequency window: .01–.1 Hz) was applied, thus for multiple comparisons (P < .05, derived from an uncor- removing linear drift artifacts and high-frequency noise. rected P < .001 on the voxel level, with a cluster extent of First-level analyses were performed correlating seed re- 69 contiguous voxels [552 mm3] as estimated by the gion signal with voxel signal throughout the whole 3dClustSim application). 3dClustSim was implemented brain, thereby creating seed region to voxel Fisher- in the Analysis of Functional Neuroimages (AFNI) soft- transformed r-to-z connectivity maps (6 maps for each ware (http://afni.nimh.nih.gov/afni/doc/manual/Alpha individual). Connectivity maps were then used for Sim, http://afni.nimh.nih.gov/afni/), based on a Monte second-level (random effects) analyses. Carlo simulation (1,000 simulations) applied to a whole Analysis 1 brain mask. Because we were specifically interested in IC-CC connectivity, a second mask, just covering the In a first step, main effects were calculated for each cingulum (anterior, middle, and posterior portions, bilat- group separately, by performing 1-sample t-tests. erally) was created using the WFU_PickAtlas (http://www. Because of the expectancy for highly correlated connec- nitrc.org/projects/wfu_pickatlas). Monte Carlo simula- tion using that mask resulted in a lower extent threshold: 28 contiguous voxels (224 mm3)(P < .001, uncorrected, on the voxel level), yielding correction for multiple compar- isons on the cluster level (within that mask).

Analysis 3a Resultant significant connectivity maps from Analysis 2 were further evaluated with correlation analyses to assess the behavioral and clinical relevance to the group differ- ences. Parameter estimates were extracted (yielding 1 parameter estimate per subject) and were correlated with pain measures (VAS, SF-MPQ affective, sensory, and total measures) and MRS pressure-pain thresholds (faint, mild and slightly intense) by means of a correlation analysis in SPSS, version 19 (SPSS, Inc, Chicago, IL). Signif- icant results were found using a threshold of P < .05, but were deemed significant at P < .025 after a Bonferroni correction accounting for the 2 domains of pain (clinical pain and experimental pain).

Analysis 3b To further evaluate the behavioral/clinical relevance of altered IC connectivity, we performed in a third step, regression analyses with pain measures (VAS, SF-MPQ af- fective, sensory, and total measures) and MRS pressure- pain thresholds as predictors and fcMRI as the outcome variable within the FM group (controlling for age). Figure 1. Figure displays the 6 seed regions used for functional connectivity analyses. Seed regions were spheres of 6 mm sur- Similarly, regression analyses with experimental pain rounding a peak voxel. Montreal Neurological Institute (MNI) measures were performed including experimental coordinates for each voxel include left anterior IC (red): pressure-pain thresholds within the HC group (control- x= 32, y = 16, z = 6; left mid IC (dark blue): x = 38, y = 2, ling for age). Again, a threshold on the cluster level z = 8; left posterior IC (green): x = 39, y = 15, z = 1; right ante- rior IC (purple): x = 32, y = 16, z = 6; right mid IC (orange): x = 38, corrected for multiple comparisons (P < .05, derived y = 2, z = 8; right posterior IC (light blue): x = 39, y = 15, z = 8. from an uncorrected P < .001 on the voxel level, with a Ichesco et al The Journal of Pain 819 cluster extent of 69 contiguous voxels) was applied. One From this analysis, we were able to partially replicate the subject was omitted from correlational analyses with the findings by Taylor et al, namely, anterior to posterior topo- SF-MPQ affective measure because of the classification of graphical connectivity between the IC and the CC. In both an outlier greater than 2 standard deviations from the patients and controls, the anterior IC was connected to the mean. The resulting clusters were further analyzed by ex- anterior CC, and the mid IC was connected to the mid CC; tracting parameter estimates from group-level results however, we were not able to replicate the posterior IC to (yielding 1 parameter estimate per subject) and perform- posterior CC connectivity in either group.43 ing the regression analyses with the pain measures in SPSS, version 19. During this analysis step, we evaluated Analysis 2 the parameter estimates to assess for outliers that could be driving the significant results. The Automated For between-group comparisons, the FM patients had Anatomical Labeling (aal) atlas45 and xjView viewing greater connectivity compared to HCs between the right program for SPM (http://www.alivelearn.net/xjview8/) anterior IC seed and the right were used to label anatomic regions for all analyses. (STG; peak voxel: x = 46, y = 26, z = 2; z-score = 4.69; Table 2), greater connectivity between the right mid IC seed and both the right mid IC (peak voxel: x = 38, Results y = 2, z = 0; z-score = 4.27, Table 2 and Fig 2), and right mid/posterior CC (peak voxel: x = 2, y = 16, z = 44; z- Patients and Behavioral Data score = 3.85, Table 2, and Fig 2). Patients also displayed All 36 subjects enrolled in this study were included in greater connectivity between the right posterior IC the analyses. Of the 18 FM patients, 7 were categorized seed and both the left mid CC (peak voxel: x = 2, to have a high level of depression (6 of the 7 FM patients y = 10, z = 30; z-score = 4.03, Table 2, and Fig 2) and the had CES-D scores >16, and the seventh scored >8 on the posterior CC (peak voxel: x = 0, y = 24, z = 46; z- Hospital Anxiety and Depression Scale). All 18 of the score = 3.88, Table 2 and Fig 2). HC subjects were assessed for depressive symptoms using Decreased connections in FM patients compared to HCs the CES-D measure—none of whom were categorized to were also found. FM patients were found to have less con- have a high level of depressive symptoms. nectivity between the left anterior IC seed and both the As expected, the FM cohort was found to have signifi- right medial frontal gyrus (peak voxel: x = 10, y = 40, cantly higher clinical pain report scores compared to the z = 40; z-score = 4.48, Table 2 and Fig 3) and the left medial HC group. However, the 2 groups did not differ statisti- frontal gyrus (peak voxel: x = 12, y = 46, z = 22; z- cally with respect to experimental pressure-pain thresh- score = 4.25; Table 2 and Fig 3), and the left posterior IC olds. Demographics and pain ratings for both patients seed and the right superior frontal gyrus (peak voxel: and controls are displayed in Table 1. x = 26, y = 20, z = 50; z-score = 4.43; Table 2 and Fig 3).

Functional Connectivity During Resting Analysis 3a State IC to CC connectivity within the FM patients was found to be related to experimental pressure-pain thresholds. Analysis 1 The right posterior IC, which showed increased connectiv- The location and size of each IC seed region is shown in ity to the posterior CC in FMs compared to HCs, was found Fig 1. Supplementary Table 1 shows the fcMRI main effect to have a strong negative correlational trend to ‘‘mild clusters for the 6 IC seed regions in the FM and HC groups pain’’ MRS pressure threshold values (r = –.50, P =.04; separately. Connectivity maps were thresholded using a Table 3). Furthermore, FM patients were found to have corrected cluster-level family-wise error value of P <.05. correlational trends between this same right posterior IC–posterior CC connectivity with the 2 other experimental pressure-pain thresholds (‘‘faint pain’’: r = –.34, P =.16;and ‘‘slightly intense pain’’: r = –.38, P = .12). Additionally, IC- Table 1. Demographics and Behavioral Data STG connectivity within the FM patients was found to be FM HC P VALUE related to clinical pain. The right anterior IC, found to Age 35.8 6 12.0 32.3 6 11.3 .375 have increased connectivity within the FM group SF-MPQ TOT 9.7 6 7.3 .3 6 .4 <.001 compared to HCs, was significantly positively correlated SF-MPQ SEN 7.2 6 5.9 .2 6 .4 <.001 to clinical pain: greater IC-STG connectivity was associated SF-MPQ AFF 2.5 6 3.3 .1 6 .3 .007 with greater affective scores from the SF-MPQ (r = .61, SF-MPQ VAS 4.4 6 2.3 .1 6 .3 <.001 P =.01;Table 3), and the total score of the SF-MPQ resulted MRS low (kg/cm2).66 .7 .8 6 .8 .505 ina strong trend (r= .51,P = .03, Table 3). Nosignificant cor- 2 MRS medium (kg/cm ) 2.3 6 1.3 2.6 6 1.4 .481 relations to clinical and experimental pain report were 2 6 6 MRS high (kg/cm ) 3.6 1.7 4.1 1.8 .347 found among the HC group within these regions. Disease duration (y) 3.9 6 3.7 – –

Abbreviations: TOT, total score; AFF, affective component; SEN, sensory compo- Analysis 3b nent. NOTE. Table includes behavioral data for all 36 subjects enrolled in this study (18 Within the FM group, the functional connectivity of the FM vs 18 HC). Values in bold were found to be statistically significant. right mid IC seed and both the left and right 820 The Journal of Pain Functional Connectivity in Fibromyalgia Table 2. Differences in Resting State Functional Connectivity Between FM and Controls

COORDINATES (MNI)

SEED REGION CONNECTIVITY REGION CLUSTER SIZE (NO. OF VOXELS) Z-SCORE (PEAK VALUE) XYZ FM > HC (ANCOVA with age as covariate of no interest) R ant IC L superior temporal gyrus 22 81 4.69 46 26 2 R mid IC R mid IC 13 81 4.27 38 2 0 R mid/post CC 24/31 57 3.85 2 16 44 R post IC L mid CC 24 123 4.03 21030 Posterior CC 31/24 40 3.88 0 24 46 HC > FM (ANCOVA with age as covariate of no interest) L ant IC R medial frontal gyrus 6 101 4.48 10 40 40 L medial frontal gyrus 9 70 4.25 12 46 22 L post IC R superior frontal gyrus 8 79 4.43 26 20 50

Abbreviations: MNI, Montreal Neurological Institute; ANCOVA, analysis of covariance; L, left; R, right. precuneus were positively correlated with the sensory Fig 4); that is, FM patients with higher sensory pain compo- component of the SF-MPQ (left precuneus: z = 3.79, nent scores had higher connectivity between the right mid P < .001; right precuneus: z = 4.48, P < .001; Table 4 and IC and both the left and right precuneus regions.

Figure 2. (A) Increased IC connectivity in FM patients compared to HCs. From left to right, increased connectivity in FM compared to HC subjects was found between the R mid IC seed and the right mid/post CC (far left), the R mid IC seed and the R mid IC (middle left), the R posterior IC seed and the L mid CC (middle left), and the R post IC seed and the posterior CC (far right). Scatter plots of connec- tivity r-value correlations for each cohort are displayed below brain figures. (B) Two graphs of the fMRI BOLD signal time series for both seed (light blue) and connected region (yellow) from a representative FM subject and an HC subject. Abbreviations: R, right; L, left; BOLD, blood oxygenation level dependent. Ichesco et al The Journal of Pain 821

Figure 3. Figure depicts decreased connectivity for FM patients when compared to HCs between the L anterior IC seed and the L medial frontal gyrus (left), the L anterior IC seed and the R medial frontal gyrus (center), and the L posterior IC seed and the R superior frontal gyrus (right). Beneath each brain image is the corresponding scatter plot of the connectivity correlations for each cohort. Ab- breviations: R, right; L, left; MFG, medial frontal gyrus; SFG, superior frontal gyrus.

FM patients were found to have several negative cor- HC subjects were only found to have 1 significant relations between experimental pain thresholds and IC correlation between connectivity and experimental connectivity: greater functional connectivity was associ- pressure-pain measures. Within this group, connectivity ated with lower pressure-pain thresholds. The left mid between the right anterior IC seed and the right poste- IC seed and the left mid CC connectivity was negatively rior CC was negatively correlated with mild pressure correlated with slightly intense pressure thresholds thresholds (r = –.53, P = .024; Table 4). (z = 3.67, P = .001; Table 4 and Fig 5); the left mid IC seed and the right mid CC connectivity was negatively correlated with faint pressure thresholds (z = 3.58; Discussion P < .001, Table 4 and Fig 5); and the right posterior IC The main objective of this work was to systematically seed and the left mid/anterior CC was negatively corre- test whether FM patients displayed regional differences lated with faint pressure thresholds (z = 4.42, P < .001; within the IC with respect to their connectivity to other Table 4 and Fig 5). brain regions compared to pain-free HCs. In a first step,

Table 3. Results of Resting State Functional Connectivity Group Differences Correlated With Clinical and Experimental Pain in FM Patients

FCMRI GROUP DIFFERENCE SEED REGION CONNECTIVITY REGION BEHAVIORAL CORRELATE P VALUE CORRELATION R FM > HC R anterior IC L superior temporal gyrus SF-MPQ AFF .01 .61* FM > HC R anterior IC L superior temporal gyrus SF-MPQ TOT .03 .51 FM > HC R posterior IC Posterior CC MRS med .04 –.50

Abbreviations: AFF, affective component; TOT, total score. NOTE. Table displays correlations between fcMRI group differences (identified in Table 2) and behavioral measures within the FM group. Correlations were deemed significant at a threshold of P < .025 based on a Bonferroni correction. Strong trends with a P < .05 are also reported. No significant correlations to experimental pain report were found among the HC group subjects within these same regions. *One subject was removed because of a response that was an outlier >2 standard deviations from the mean. 822 The Journal of Pain Functional Connectivity in Fibromyalgia Table 4. Results of Behavioral Correlations With Whole Brain Resting State Functional Connectivity

COORDINATES (MNI)

SEED REGION CONNECTIVITY REGION BEHAVIORAL CORRELATE BA CLUSTER SIZE (NO. OF VOXELS) Z-SCORE XYZ FM, positive correlations R mid IC R precuneus SF-MPQ SEN 19 136 4.48 28 78 42 R SMA SF-MPQ AFF* 6 80 4.03 24 14 62 L precuneus SF-MPQ SEN 7 71 3.79 6 54 50 FM, negative correlations L mid IC L mid CC MRS high 24 29 3.67 4 248 R mid CC MRS low 24 56 3.58 4 4 40 R mid IC Mid CC SF-MPQ AFF* 24 42 4.12 0 10 48 L posterior IC SF-MPQ AFF* 13 45 3.97 42 42 R posterior IC L precuneus MRS low 19 223 4.74 6 76 38 L mid/anterior IC MRS low 13 42 4.42 40 6 12 R STG MRS low 13/22 95 4.19 54 42 18 HC, positive correlations R anterior IC R posterior CC MRS medium 31 57 4.20 10 32 44

Abbreviations: MNI, Montreal Neurological Institute; R, right; L, left; SEN, sensory component; SMA, ; AFF, affective component. *One subject was removed because of a response that was an outlier >2 standard deviations from the mean.

we were able to comparatively reproduce the results by FM patients, whereas other regions showed increased Taylor et al showing topographically distributed and connectivity in the controls. Largely, these findings highly correlated low-frequency oscillations between showed that patients had increased connectivity of the specific IC and CC subdivisions43 (ie, IC-CC connectivity right insula whereas controls had increased connectivity was detected along the anterior-middle axis) in both of the left insula. Previous studies have shown that there HCs and FM patients. These findings are also in line is lateralized cerebral function with regard to the pro- with a study by Kong et al reporting resting-state fcMRI cessing of pain, where the right hemisphere has been between the anterior IC and the ACC/MCC in healthy shown to have more involvement in pain perception subjects.24 Interestingly, our group comparison between and sensitivity compared to the left hemisphere.28,33,40 the FM and HC groups yielded significantly increased Furthermore, Kucyi et al reported right hemisphere connectivity between the IC and CC regions within the lateralization in intrinsic functional connectivity

Figure 4. Figure displays connectivity in FM patients between the IC and precuneus. Connectivity between the R mid IC seed and the L precuneus was positively associated with SF-MPQ sensory pain report (top). Also, connectivity between the R mid IC seed and the R precuneus was positively associated with the SF-MPQ sensory pain report (bottom). In both cases, this result shows that FM patients display increased pain report as the IC-precuneus connectivity increases. Ichesco et al The Journal of Pain 823

Figure 5. Figure displays connectivity in FM patients between the IC and mid CC and IC. Connectivity between the L mid IC seed and the L MCC was negatively associated with ‘‘slightly intense’’ pressure thresholds (top). Also, connectivity between the L mid IC seed and the R MCC was negatively associated with ‘‘faint’’ pressure thresholds (middle). Finally, connectivity between the R posterior IC seed and the left mid IC was negatively correlated with ‘‘faint’’ pressure thresholds (bottom). In all cases, this result shows that FM patients display decreased experimental pain thresholds as IC-CC and IC-IC connectivity increases. Abbreviations: L, left; R, right.

between the , a brain region associated with increased connectivity between these re- involved in salient stimulus detection, and the right gions. Moreover, we found additional negative correla- mid IC.25 In this study, we found augmented right hemi- tions between experimental pain and IC connectivity to sphere lateralization resting-state connectivity including nociceptive regions. These results included increased the IC in FM patients compared to HCs. The chronifica- connectivity between the bilateral mid IC seed regions tion process within these patients very well could have and the mid CC and contralateral posterior IC target re- strengthened this relationship in the right hemisphere gions (ie, IC-CC and IC-IC connectivity), which corre- and caused this increased connectivity of pronociceptive sponded with decreased experimental pressure-pain regions while at rest. Additionally, when comparing cor- thresholds within FM patients. relation measures between the FM and HC groups, it ap- Insular involvement in processing and modulation of pears that FM patients have formed significant new pain has been widely reported as it is one of the most connections between the right IC and CC regions (Fig commonly activated pain regions in neuroimaging 2), and added deficits between the left IC and medial studies of pain.1 Previously, FM patients were reported frontal gyrus regions (Fig 3). to have increased IC connectivity when compared to HC When comparing our correlational findings with subjects, and FM patients were found to have positive experimental pain reports, we found strong negative as- correlations between current clinical pain intensities sociations between posterior IC–posterior CC connectiv- and intrinsic connectivity between the IC and both ity and experimental pain thresholds in pain patients. DMN and Executive Attention Network.30,31 It has been Because pain thresholds are effectively the inverse of suggested that bilateral IC activation is essential for pain sensitivity, these findings are showing that pain perception, as opposed to nonpainful increased pain sensitivity (ie, hyperalgesia/allodynia) is somatosensory perception. In 2007, Seifert and 824 The Journal of Pain Functional Connectivity in Fibromyalgia Maihofner€ displayed bilateral IC activation in a sample of have increased connectivity between the IC and the HC subjects when undergoing an fMRI scan with a cold DMN at rest, and that this connectivity is associated allodynia stimulus application.41 A more recent study with increased current clinical pain.31 The current of HC subjects that used arterial spin labeling reported finding appears to support this finding that patients results of increased cerebral blood flow to the bilateral with FM have hyperactive IC-DMN connectivity and IC in response to increased intramuscular infusion of increased associated clinical pain as previously reported hypertonic saline into the brachioradialis muscle.32 by our group.30,31 Within HCs, high connectivity has been displayed Interestingly, although not an a priori hypothesis, we between the IC and the contralateral insula during a noted that FM patients were found to have increased resting state.5,43 Here, FM patients were found to have connectivity between the right anterior insula and the increased resting-state fcMRI compared to HC contralateral STG. This heightened connectivity result volunteers. This amplified connectivity between the for the FM patients was found to be positively correlated insular regions in these FM patients could contribute to with clinical pain (increased connectivity was associated the widespread nonarticular pain that is symptomatic with increased pain scores)—with both the affective (sig- of this disorder. nificant) and total (trend) scores from the SF-MPQ. The It has been suggested that the anterior IC-ACC system role of the STG in pain is not well established, but this re- integrates interoceptive input with its emotional gion has been shown to be active during evoked painful in contrast to the mid/posterior IC-MCC system, stimuli in both HC and FM patients.17 Although the which is thought to be more related to environmental mechanism of the STG in pain processing may not be fully monitoring and response selection.43 With respect to understood, it is possible that this region could be pain perception, there is strong evidence that the poste- contributing to the augmented affective experience of rior IC, as part of the lateral pain pathway, together with pain in FM patients, as we have found this increased the primary and secondary somatosensory cortices, en- anterior IC to STG connectivity to be positively correlated codes pain intensity, laterality, and somatotopy, whereas with affective pain report scores. Furthermore, FM pa- the anterior IC, as part of the medial pathway, together tients were found to have a negative correlation with the ACC, has a unique role in affective pain process- displaying increased connectivity between the posterior ing and learning.44 IC and STG, with decreases in experimental pressure-pain Although there is an increasing body of evidence that thresholds (faint pain). suggests that the IC flexibly connects attention and emotional brain areas, and that these connections are in fact an important determinant of pain experience,38 Limitations the literature on the capability of the mid CC to modu- Although our trial found significant differences in late IC activity in pain conditions, or vice versa, is sparse. IC-CC connectivity in FM, there are certain limitations In this study, we observed augmented mid/posterior IC– that need to be highlighted. One possible confounder mid/posterior CC connectivity within FM patients in our study was age. Although not statistically compared to HCs. Moreover, these regions with different, the FM group was slightly older than the increased IC-CC connectivity displayed correlations with HCs. To control for this potential confound, age was experimental pressure-pain thresholds. FM patients added as a covariate to all second-level analyses. were found to have decreased pressure-pain tolerances Another limitation in our study was the cross- (slightly intense pain) and thresholds (faint pain) when sectional design and its inability to resolve conclusively displaying increased mid IC–mid CC connectivity as well the preexisting versus acquired nature of the observed as posterior IC–mid IC connectivity. When this compari- alterations. From our results, it is unclear whether son was assessed in HC subjects, no such correlation ex- chronic widespread pain leads to the changes in con- isted. Although we did not see significant behavioral nectivity or if changes in IC connectivity predispose differences in experimental pressure pain between someone to developing chronic pain. Another poten- groups in this study, this correlation result falls in line tial weakness of our design is that we used standard- with differences in experimental pressure-pain thresh- ized seed regions. Subtle albeit natural differences in olds between HC and FM patients seen in other studies,35 functional anatomy across subjects and differences in and associates the sensation of pain with the underlying brain size and subsequent normalization might have neurobiological mapping within these FM patients. had an influence on connectivity maps. However, we Another intriguing result from an a priori hypothesis would assume that variation in functional anatomy is was that FM patients were found to have IC to DMN re- equally distributed between groups, and the fact gion connectivity correlated with clinical pain report. that images had been smoothed prior to analysis Within the FM cohort, increased connectivity between helped to control for such differences. Indeed we the right mid IC and bilateral precuneus regions was were able to partially replicate the findings of Taylor positively associated with the sensory aspect of pain (ie, et al, suggesting consistent findings.43 A minor limita- the stronger the connectivity between these 2 regions, tion for this study was that when confirming handed- the higher the level of pain reported). The precuneus is ness, a validated tool like the Edinburgh Handedness a region that contributes to the DMN, which is disen- Inventory was not included. Although all participants gaged during a variety of task conditions.4,14 reported they were right-handed, an instrument to Previously, our group reported that patients with FM confirm this measure would prove useful for validation Ichesco et al The Journal of Pain 825 in an fcMRI study. Finally, fcMRI allows no assumptions brain in individuals with FM as compared to HCs. Our an- on causality, or on directedness of influence; namely, alyses reveal increased IC-CC and IC-IC connectivity in FM, we cannot tell if the IC is sending pain information and in addition we find both positive and negative rela- to the CC or vice versa. Against this background, it is tionships between IC connectivity to other brain regions conceivable that fcMRI between 2 regions may also and multiple pain domains. These findings are indicative be driven by a third region not identified in the anal- of a disturbed IC connectivity in FM patients that may be ysis. More sophisticated approaches exploring effec- related to the subjective enhanced pain that they experi- tive connectivity and the relationship between ence. functional and structural connectivity12 will help to overcome such methodological shortcomings in future Acknowledgment studies. We would like to thank Keith Newnham for MRI tech- Conclusions and Outlook nical assistance. The investigation of resting-state networks is a prom- ising approach and might in fact turn out to be a stron- ger tool than approaches using evoked pain paradigms when it comes to the exploration of subjective states Supplementary Data such as clinical pain. Our main goal was to explore Supplementary data related to this article can be regional differences in IC connectivity to the rest of the found at http://dx.doi.org/10.1016/j.jpain.2014.04.007.

References and resting-state functional connectivity. Brain Struct Funct 213:525-533, 2009

1. Apkarian AV, Bushnell MC, Treede RD, Zubieta JK: Human 13. Davis KD, Taylor KS, Hutchison WD, Dostrovsky JO, brain mechanisms of pain perception and regulation in McAndrews MP, Richter EO, Lozano AM: Human anterior health and disease. Eur J Pain 9:463-484, 2005 cingulate cortex neurons encode cognitive and emotional demands. J Neurosci 25:8402-8406, 2005 2. Birn RM: The behavioral significance of spontaneous fluc- tuations in brain activity. Neuron 56:8-9, 2007 14. Fox MD, Raichle ME: Spontaneous fluctuations in brain activity observed with functional magnetic resonance imag- 3. Birn RM, Diamond JB, Smith MA, Bandettini PA: Sepa- ing. Nat Rev Neurosci 8:700-711, 2007 rating respiratory-variation-related fluctuations from neuronal-activity-related fluctuations in fMRI. Neuroimage 15. Gracely RH, Kwilosz DM: The Descriptor Differential 31:1536-1548, 2006 Scale: Applying psychophysical principles to clinical pain assessment. Pain 35:279-288, 1988 4. Buckner RL, Vincent JL: Unrest at rest: Default activity and spontaneous network correlations. Neuroimage 37: 16. Gracely RH, Lota L, Walter DJ, Dubner R: A multiple 1091-1096, 2007. discussion 1097-9 random staircase method of psychophysical pain assess- ment. Pain 32:55-63, 1988 5. Cauda F, D’Agata F, Sacco K, Duca S, Geminiani G, Vercelli A: Functional connectivity of the insula in the resting 17. Gracely RH, Petzke F, Wolf JM, Clauw DJ: Functional brain. Neuroimage 55:8-23, 2011 magnetic resonance imaging evidence of augmented pain processing in fibromyalgia. Arthritis Rheum 46:1333-1343, 6. Cauda F, Sacco K, D’Agata F, Duca S, Cocito D, 2002 Geminiani G, Migliorati F, Isoardo G: Low-frequency BOLD fluctuations demonstrate altered thalamocortical connec- 18. Greicius MD, Krasnow B, Reiss AL, Menon V: Functional tivity in diabetic neuropathic pain. BMC Neurosci 10:138, connectivity in the resting brain: A network analysis of the 2009 default mode hypothesis. Proc Natl Acad Sci U S A 100: 253-258, 2003 7. Chang C, Glover GH: Effects of model-based physiological noise correction on default mode network anti-correlations 19. Harris RE, Gracely RH, McLean SA, Williams DA, and correlations. Neuroimage 47:1448-1459, 2009 Giesecke T, Petzke F, Sen A, Clauw DJ: Comparison of clinical and evoked pain measures in fibromyalgia. J Pain 7:521-527, 8. Cook DB, Lange G, Ciccone DS, Liu WC, Steffener J, 2006 Natelson BH: Functional imaging of pain in patients with primary fibromyalgia. J Rheumatol 31:364-378, 2004 20. Harris RE, Napadow V, Huggins JP, Pauer L, Kim J, Hampson J, Sundgren PM, Foerster B, Petrou M, Schmidt- 9. Craig AD: How do you feel? : The of Wilcke T, Clauw DJ: Pregabalin rectifies aberrant brain the physiological condition of the body. Nat Rev Neurosci chemistry, connectivity, and functional response in chronic 3:655-666, 2002 pain patients. Anesthesiology 119:1453-1464, 2013

10. Craig AD: How do you feel–now? The anterior insula 21. Hu X, Le TH, Parrish T, Erhard P: Retrospective estimation and human awareness. Nat Rev Neurosci 10:59-70, 2009 and correction of physiological fluctuation in functional MRI. Magn Reson Med 34:201-212, 1995 11. Critchley HD, Wiens S, Rotshtein P, Ohman A, Dolan RJ: Neural systems supporting interoceptive awareness. Nat 22. Ichesco E, Quintero A, Clauw DJ, Peltier S, Sundgren PM, Neurosci 7:189-195, 2004 Gerstner GE, Schmidt-Wilcke T: Altered functional connec- tivity between the insula and the cingulate cortex in pa- 12. Damoiseaux JS, Greicius MD: Greater than the sum of its tients with temporomandibular disorder: A pilot study. parts: A review of studies combining structural connectivity Headache 52:441-454, 2012 826 The Journal of Pain Functional Connectivity in Fibromyalgia 23. Ichesco E, Schmidt-Wilcke T, Clauw D, Peltier S, off-resonance effects in dynamic echo-planar and spiral Williams D, Harris R: Altered resting connectivity between functional imaging. Magn Reson Med 47:344-353, 2002 the insula and cingulate cortex is related to chronic fibromy- algia pain. J Pain 13:S29, 2012 38. Ploner M, Lee MC, Wiech K, Bingel U, Tracey I: Flexible cerebral connectivity patterns subserve contextual modula- 24. Kong J, Loggia ML, Zyloney C, Tu P, Laviolette P, tions of pain. Cereb Cortex 21:719-726, 2011 Gollub RL: Exploring the brain in pain: Activations, deactiva- tions and their relation. Pain 148:257-267, 2010 39. Radloff LS: The CES-D Scale: A self-report depression scale for research in the general population. Appl Psychol 25. Kucyi A, Hodaie M, Davis KD: Lateralization in intrinsic Meas 1:385-401, 1977 functional connectivity of the temporoparietal junction with salience- and attention-related brain networks. J Neu- 40. Sarlani E, Farooq N, Greenspan JD: Gender and laterality rophysiol 108:3382-3392, 2012 differences in thermosensation throughout the perceptible range. Pain 106:9-18, 2003 26. Limongi R, Sutherland SC, Zhu J, Young ME, Habib R: € Temporal prediction errors modulate cingulate-insular 41. Seifert F, Maihofner C: Representation of cold allodynia coupling. Neuroimage 71:147-157, 2013 in the —A functional MRI study. Neuroimage 35:1168-1180, 2007 27. Loggia ML, Kim J, Gollub RL, Vangel MG, Kirsch I, Kong J, Wasan AD, Napadow V: Default mode network connectivity 42. Spielberger CD. http://mindgarden.com/products/stpi. encodes clinical pain: An arterial spin labeling study. Pain htm. Accessed March 18, 2010 154:24-33, 2013 43. Taylor KS, Seminowicz DA, Davis KD: Two systems of 28. Lugo M, Isturiz G, Lara C, Garcia N, Eblen-Zaijur A: Sen- resting state connectivity between the insula and cingulate sory lateralization in pain subjective perception for noxious cortex. Hum Brain Mapp 30:2731-2745, 2009 heat stimulus. Somatosens Mot Res 19:207-212, 2002 44. Tracey I: Nociceptive processing in the human brain. Curr 29. Melzack R: The Short-Form McGill Pain Questionnaire. Opin Neurobiol 15:478-487, 2005 Pain 30:191-197, 1987 45. Tzourio-Mazoyer N, Landeau B, Papathanassiou D, 30. Napadow V, Kim J, Clauw DJ, Harris RE: Decreased Crivello F, Etard O, Delcroix N, Mazoyer B, Joliot M: intrinsic brain connectivity is associated with reduced clin- Automated anatomical labeling of activations in SPM ical pain in fibromyalgia. Arthritis Rheum 64:2398-2403, using a macroscopic anatomical parcellation of the 2012 MNI MRI single-subject brain. Neuroimage 15:273-289, 2002 31. Napadow V, LaCount L, Park K, As-Sanie S, Clauw DJ, Harris RE: Intrinsic brain connectivity in fibromyalgia is asso- 46. Vogt BA: Pain and interactions in subregions of ciated with chronic pain intensity. Arthritis Rheum 62: the cingulate gyrus. Nat Rev Neurosci 6:533-544, 2005 2545-2555, 2010 47. Whitfield-Gabrieli S, Nieto-Castanon A: Conn: A func- 32. Owen DG, Clarke CF, Bureau Y, Ganapathy S, Prato FS, St tional connectivity toolbox for correlated and anticorre- Lawrence KS: Measuring the neural response to continuous lated brain networks. Brain Connect 2:125-141, 2012 intramuscular infusion of hypertonic saline by perfusion MRI. J Magn Reson Imaging 35:669-677, 2012 48. Williams DA, Clauw DJ: Understanding fibromyalgia: Lessons from the broader pain research community. J Pain 33. Pauli P, Wiedemann G, Nickola M: Pain sensitivity, cere- 10:777-791, 2009 bral laterality, and negative affect. Pain 80:359-364, 1999

34. Petzke F, Clauw DJ, Ambrose K, Khine A, Gracely RH: 49. Williams DA, Schilling S: Advances in the assessment of Increased pain sensitivity in fibromyalgia: Effects of stimulus fibromyalgia. Rheum Dis Clin North Am 35:339-357, 2009 type and mode of presentation. Pain 105:403-413, 2003 50. Wolfe F, Smythe HA, Yunus MB, Bennett RM, 35. Petzke F, Harris RE, Williams DA, Clauw DJ, Gracely RH: Bombardier C, Goldenberg DL, Tugwell P, Campbell SM, Differences in unpleasantness induced by experimental Abeles M, Clark P, Fam AG, Farber SJ, Fiechtner JJ, pressure pain between patients with fibromyalgia and Franklin CM, Gatter RA, Hamaty D, Lessard J, healthy controls. Eur J Pain 9:325-335, 2005 Lichtbroun AS, Masi AT, Mccain GA, Reynolds WJ, Romano TJ, Russell IJ, Sheon RP: The American College of 36. Peyron R, Laurent B, Garcia-Larrea L: Functional imaging Rheumatology 1990 Criteria for the Classification of Fibro- of brain responses to pain. A review and meta-analysis myalgia. Report of the Multicenter Criteria Committee. (2000). Neurophysiol Clin 30:263-288, 2000 Arthritis Rheum 33:160-172, 1990 37. Pfeuffer J, Van de Moortele PF, Ugurbil K, Hu X, 51. Zigmond AS, Snaith RP: The hospital anxiety and depres- Glover GH: Correction of physiologically induced global sion scale. Acta Psychiatr Scand 67:361-370, 1983 Ichesco et al 826.e1 Supplementary Table 1. Main Effect of Resting State Functional Connectivity

COORDINATES (MNI)

SEED REGION CONNECTIVITY REGION BRODMANN AREA CLUSTER SIZE (NO. OF VOXELS) Z-SCORE (PEAK VALUE) XYZ FM L anterior IC L anterior IC 13/22 2,414 Inf 32 16 8 R anterior IC 13/22 1,364 6.31 36 16 6 R 40 146 5.76 52 30 20 R anterior/mid CC 24/32 167 5.60 10 16 32 L inferior parietal lobule 40 298 5.27 60 42 42 Supplementary motor area 32/6/24 81 5.10 0 8 48 L mid IC L mid IC 13 3,505 Inf 36 2 6 R mid/posterior IC 13 2,296 6.51 36 8 12 L/R mid CC/SMA 24//6/32 628 5.65 21230 L posterior IC L posterior IC 13 2,027 Inf 40 14 12 R posterior IC 13 615 5.53 42 12 14 R anterior IC R anterior IC 13/44 2,095 Inf 34 16 6 L anterior IC 13 1,487 6.47 28 14 8 L inferior parietal lobule 40 160 5.67 58 42 42 L anterior CC 32/24 442 5.64 22428 R inferior parietal lobule 40 276 5.58 50 36 30 L DLPFC 9 72 5.44 36 32 32 R DLPFC 10/46 248 5.33 40 36 28 R mid IC R mid IC 13 4,378 Inf 38 2 6 L mid IC 13 3,031 6.70 34 10 4 L mid CC/SMA 24/6/32 522 6.05 2446 R DLPFC 10/9 140 5.20 40 44 24 R posterior IC R posterior IC 13/22 2,517 Inf 40 16 10 L posterior IC 13 1,029 6.01 50 16 6 HC L anterior IC L anterior IC 13/44 1,412 Inf 32 16 8 R anterior IC 13 294 5.68 30 18 2 L mid front gyrus/DLPFC 10/9 240 5.54 42 40 30 L/R mid CC/SMA 32 611 5.42 2852 R DLPFC 46 79 5.34 42 40 24 L inferior parietal lobule 40 85 5.10 58 30 28 L mid IC L mid IC 13/22 3,179 Inf 38 4 8 R mid IC 13/22 1,429 6.43 52 10 0 L/R mid CC/SMA 32/6/24 781 5.81 2 10 46 R STG/IPL 40/22 514 6.06 30 40 26 L posterior IC L posterior IC 13 1,887 Inf 40 16 10 R posterior IC 13 462 5.81 42 14 0 R anterior IC R anterior IC 13/22 1,259 Inf 32 16 4 L/R anterior/mid CC 32/24 619 5.91 2244 L anterior IC 13 286 5.49 34 18 8 R DLPFC 10/46 102 5.00 40 40 20 R mid IC R mid IC 13/22 2,372 Inf 38 2 8 L posterior/mid IC 13/22 1,065 5.78 48 46 R mid CC/SMA 32/24/6 491 5.67 2452 L inferior parietal lobule 40 228 5.57 48 26 14 R posterior IC R posterior IC 13 1,485 Inf 40 14 8 L posterior IC 13/22 741 5.57 60 20

Abbreviations: MNI, Montreal Neurological Institute; L, left; R, right; SMA, supplementary motor area; DLPFC, dorsolateral ; IPL, inferior parietal lobule; Inf, infinite. NOTE. Table displays main effect connectivity between the insula seed regions and significantly connected regions for both the FM and HC groups. Connectivity map results were deemed significant at a cluster-level corrected P < .05 derived from an uncorrected P < .00001 on the voxel level.