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NeuroImage 41 (2008) 1032–1043

Sex differences in brain activity during aversive visceral stimulationand its expectation in patients with chronic abdominal pain:A network analysis☆

J.S. Labus,a,c,d,⁎ B.N. Naliboff,a,c,e J. Fallon,g S.M. Berman,a,b,c,d B. Suyenobu,a,b

J.A. Bueller,a,b M. Mandelkern,a,g and E.A. Mayera,b,c,d

aCenter for Neurobiology of Stress, David Geffen School of Medicine at UCLA, USAbDepartment of Medicine, David Geffen School of Medicine at UCLA, USAcDepartment of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine at UCLA, USAdBrain Research Institute, David Geffen School of Medicine at UCLA, USAeVA Greater Los Angeles Healthcare System, Los Angeles, CA 90073, USAfDepartment of Anatomy and Neurobiology, UC Irvine, Irvine, CA 92697, USAgDepartment of Physics, UC Irvine, Irvine, CA 92697, USA

Received 18 November 2007; revised 23 February 2008; accepted 3 March 2008Available online 20 March 2008

Differences in brain responses to aversive visceral stimuli may underliepreviously reported sex differences in symptoms as well as perceptualand emotional responses to such stimuli in patients with irritable bowelsyndrome (IBS). The goal of the current study was to identify brainnetworks activated by expected and delivered aversive visceral stimuliin male and female patients with chronic abdominal pain, and to testfor sex differences in the effective connectivity of the circuitrycomprising these networks. Network analysis was applied to assessthe brain response of 46 IBS patients (22 men and 24 women) recordedusing [15O] water positron emission tomography during rest/baselineand expected and delivered aversive rectal distension. Functionalconnectivity results from partial least squares analyses providedsupport for the hypothesized involvement of 3 networks correspondingto: 1) visceral afferent information processing (thalamus, insula anddorsal anterior cingulate cortex, orbital frontal cortex), 2) emotional-arousal (amygdala, rostral and subgenual cingulate regions, and locuscoeruleus complex) and 3) cortical modulation (frontal and parietalcortices). Effective connectivity results obtained via structural equationmodeling indicated that sex-related differences in brain response arelargely due to alterations in the effective connectivity of emotional-

☆ Supported in part by grants from the National Institutes of Health (K08DK071626 (JSL), P50 DK064539 (EAM), RO1 DK 48351(EAM)), theOffice of Research in Women's Health (ORWH) (P50 DK064539 (EAM))and the National Center for Complementary and Alternative Medicine(NCCAM) (R24 AT002681).⁎ Corresponding author. Center of Neurovisceral Sciences and Women's

Health, Neuroimaging Imaging Core, Peter V. Ueberroth Building, Rm2338C2, 10945 LeConte Avenue, Los Angeles, CA 90095, USA. Fax: +1310 825 1919.

E-mail address: jlabus@ucla.edu (J.S. Labus).Available online on ScienceDirect (www.sciencedirect.com).

1053-8119/$ - see front matter © 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.neuroimage.2008.03.009

arousal circuitry rather than visceral afferent processing circuits. Sexdifferences in the cortico-limbic circuitry involved in emotional-arousal, pain facilitation and autonomic responses may underlie theobserved differences in symptoms, and in perceptual and emotionalresponses to aversive visceral stimuli.© 2008 Elsevier Inc. All rights reserved.

Introduction

Irritable bowel syndrome (IBS) is one of the most commonfunctional pain disorders, characterized by chronic abdominal painor discomfort (related to visceral hyperalgesia) associated withaltered bowel habits (related to autonomic dysregulation). Affectedpatients show frequent comorbidity with anxiety, (Mayer et al.,2001) as well as symptom-related anxiety (e.g. fears and worryover expected abdominal pain and discomfort (Labus et al., 2007)).Altered brain gut interactions during a visceral stimulus and itsexpectation have been suggested as an important pathophysio-logical mechanism for IBS, and functional brain imaging studieshave identified brain regions and circuits which may be responsiblefor these alterations (Mayer et al., 2006).

Like many other syndromes characterized by chronic physical oremotional pain and discomfort, IBS is significantly more common inwomen (Chang and Heitkemper, 2002) and sex-related differencesin the perceptual and emotional responses of IBS patients to aversivevisceral stimuli have been reported (Chang et al., 2006b; Heitkemperet al., 2003; Mayer et al., 2004; Tillisch et al., 2005). Greatersubjective responses in female IBS patients may be related to sexdifferences in brain responses to visceral stimuli (Berman et al.,

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2000, 2006; Naliboff et al., 2003). For example, female patientsshowed greater activation of limbic and paralimbic regions,including the amygdala and the closely connected anterior cingulatecortex (ACC) while male patients demonstrated greater activation ofthe insula (INS). Although activation analyses have suggestedpossible regional differences in central processing between healthycontrols and IBS, and between male and female IBS patients(reviewed in Mayer et al., 2006), they are limited in their ability todescribe more complete system-level models of the functionalneurocircuitry that may be involved.

The current study applied network analyses to test the generalhypothesis that at least 3 networks can be identified as operatingduring an expected and a delivered aversive visceral stimulusincluding: a) a network central to processing of visceral afferentinformation (“homeostatic-afferent” network i.e., thalamus, posterior(p) and anterior (a) INS and dorsal (d) ACC, orbital frontal cortex(OFC) (Craig, 2003b,c; Mayer et al., 2006)), b) a network involved inarousal, and emotion-related pain amplification (“emotional-arousal”network i.e., amygdala, ACC subregions and locus coeruleuscomplex (LCC) (Pezawas et al., 2005; Stein et al., 2007; Valentinoet al., 1999)), and c) a network representing themediating influence ofcortical regions (“cortical-modulatory” network i.e., frontal, parietal)on a) and b) (Mayer et al., 2005; Naliboff et al., 2006). Even thoughthese networks overlap and share some of the same regions, wedecided to reduce the complexity of the model and discuss the 3networks separately.

Based on the proposed role of the amygdala in cognitive andaffective modulation of pain (Carrasquillo and Gereau, 2007;Neugebauer et al., 2004) and consistent reports of sex-relateddifferences in amygdala responses in healthy subjects (Cahill, 2006)and IBS patients (Naliboff et al., 2003), we further hypothesized thatmost of the sex-related differences in brain response are in theeffective connectivity of the “emotional-arousal” network, and lessin the “cortical-modulatory” and “homoeostatic-afferent” networks.Specifically, we tested the following hypotheses regarding theactivity within nodes, and the connectivity between nodes withinthe 3 networks: 1) Across conditions, male and female IBS patientsshow similar activity/connectivity in the “homeostatic-afferent”processing network. 2) Across conditions, the activity/connectivityof amygdala-related network(s) shows sex differences. 3) There aresex-related differences in the effective connectivity of the “emotional-arousal” network during both conditions. Specifically, we expectedfemale patients to show greater activity/connectivity or engagementof the amygdala-related networks. Parts of these results have beenpublished in abstract form (Labus et al., 2005, 2006).

Methods

Data from a previously published [15O] water positron emissiontomography (PET) neuroimaging study (Naliboff et al., 2003) wereanalyzed. The sample included 46 men (n=22) and women (n=24)with a diagnosis of IBS (Rome I criteria) (Thompson et al., 1994).All patients were free from centrally acting medication for at least30 days preceding the PETscan. Patients had no history of substanceabuse or psychiatric illness. On average, women were 41.5 (10.8)years old and reported a 6month symptom severity of 13.4 (2.5) on a20-cm Verbal Descriptor Visual Analogue Scale (VDVAS). Menwere 39.8 (10.8) years old and reported similar levels of symptomseverity (12.7 (2.6)). Women had more psychological symptomsthan men (57.6 (9.8) vs. 49.2 (10.6), pb .01) as assessed by theGeneral Severity Index of the SCL90-R questionnaire (Derogatis,

1983). However, means for both men and women were below theclinically significant cut-off scores.

The experimental protocol has been described in detail previously(Naliboff et al., 2003). Briefly, PET counts (Siemens/CTI 953tomograph, Siemens-Computer Technology, Knoxville TN) from 31contiguous axial planes were summed over 90 s after threeintravenous administrations of 25 mCi [15O] water to constructvolume images reflecting regional cerebral blood flow (rCBF) during1) an initial resting baseline period (BL), 2) an expected moderate(45 mmHg) rectal balloon distension (INF), and finally 3) anexpected but undelivered distension (EXP). In the original study,these 3 conditions were repeated following a train of repeatedsigmoid colon distensions. Data analyzed in the current study wereonly taken from the initial 3 conditions, prior to the sigmoid stimulus.During the INF scan, the rectal balloon was inflated to 45 mmHgpressure for 60 s. This pressure has been shown to be associated witha subjective rating of discomfort rather than pain (Posserud et al.,2007). Men and women rated this stimulus at similar levels ofintensity on a 20-cm VDVAS (mean (SD) 11.9 (3.2) and 12.3 (3.0),respectively). Intensity ratings for EXP were also similar betweenmen (3.8 (4.1)) and women (3.2 (3.6)). All scans were preprocessed(SPM99, Wellcome Trust Centre for the Study of CognitiveNeurology, London, UK) by realignment to the initial scan for eachsubject, registration into the standardized space of the average MRIbrain image provided by the Montreal Neurological Institute (MNIspace), spatial smoothing with an isotropic 12 mm FWHMGaussianfilter, and reslicing to 4 mm isotropic voxels.

Statistical analysis

Overview

Statistical analyses were performed in steps that will be detailedbelow. First, a multivariate task partial least squares (PLS) (McIntoshet al., 1996, 2004; McIntosh and Lobaugh, 2004) identified spatiallydistributed patterns of regions activated during INF and EXP relativeto BL in males and females. These results, in combination withrelevant theoretical and neurobiological information from earlierstudies, indicated that bilateral amygdala, and thalamuswere involvedin the brain's response during EXP, and detection of aversive visceralstimuli, respectively.

Consistent reports of sex-related differences in amygdalaresponses have been reported during anticipation of aversiveemotional stimuli in healthy subjects (Buchel et al., 1998; Cahill,2006; Mackiewicz et al., 2006; Sarinopoulos et al., 2006) and IBSpatients (Berman et al., 2008; Naliboff et al., 2003) and amygdalaactivity is associated with the cognitive and affective modulation ofpain (Carrasquillo and Gereau, 2007; Neugebauer et al., 2004).

Several studies have demonstrated thalamic activation duringrectal distension in healthy controls and IBS patients (Baciu et al.,1999; Kwan et al., 2005; Mertz et al., 2000; Naliboff et al., 2001;Ringel et al., 2003; Silverman et al., 1997; Verne et al., 2003; Wilder-Smith et al., 2004; Yuan et al., 2003). Indeed greater activations of thethalamus have been shown with visceral as compared to somaticstimuli in IBS (Verne et al., 2003). Visceral afferent informationascends in the lamina I spino-thalamic tract to a specific thalamo-cortical relay nucleus (VMpo) in posterolateral thalamus, which inturn projects to a discrete portion of dorsal posterior insular cortex.This ascending afferent lamina I pathway in primates and humans alsoprovides a direct thalamo-cortical pathway (by way of MDvc inmedial thalamus) that activates the dorsal anterior cingulate cortex.

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A seed PLS was applied to test for group- and condition-specific differences in the functional connectivity of the amygdalaand thalamus with the rest of the brain (whole-brain activity).Finally, the effective connectivity of a hypothesized neural networkcomprised of “emotional-arousal,” “homeostatic-afferent”, and“cortical-modulatory” circuitry was specified and tested for sexdifferences using structural equation modeling.

Partial least squares analyses

PLS is a multivariate statistical technique that is analogous toprincipal components analysis (PCA), but the solutions are restrictedto the part of the covariance structure that is attributable to conditionsor groups in an experimental design (task PLS), behavioral measures(behavioral PLS), or the activity within a specific brain region(seed PLS). Behavioral and seed PLS are functional connectivityanalyses that provide a test for hypotheses regarding the brain regionscomprising neural networks. PLS analyses were implementedusing freely available code (http://www.rotman-baycrest.on.ca) inMatlab7.01 (Mathworks, Natick, MA).

Task PLSFirst, a “nonrotated” task PLS extracted spatial patterns where

brain activity changed during INF and EXP relative to the BL scan.The transpose of a matrix comprising four a priori nonorthogonalexperimental design contrasts (main effects of condition: BL-INF,BL-EXP, and the interaction of multiplied times sex with each maineffect) was mutliplied with a data matrix containing the normalizedsignal intensity measure at each voxel (i.e., rCBF adjusted for globalCBF by dividing each voxel by the mean voxel activity of the whole-brain image) to produce a “cross-block covariance” matrix compris-ing four latent variables (LVs) or spatial patterns of brain activityrelated to the specified design contrasts. The data matrix comprised138 (46 subjects×3 conditions) rows and one column for each voxel.The design matrix comprised 138 rows×4 columns for the designcontrasts. Unlike its “rotated” counterpart, a “nonrotated” PLS doesnot employ singular value decomposition (SVD) as an exploratorytool to extract LVs corresponding to experimental patterns (e.g.,design contrasts) accounting for themaximum amount of independentvariance in the data. Instead, the columns of the “cross-blockcovariance” matrix are considered the brain LVs and the squaredsingular value for eachLVis the sumof the squares of the “cross-blockcovariance” matrix column (McIntosh et al., 1996; McIntosh andLobaugh, 2004).

The significance of each LV was assessed via nonparametricpermutation testing using 500 permutations. This resamplingtechnique, which reassigns the order of the conditions for eachobservation, determines whether the effect represented by the LV issignificantly different from random noise and is thought to approxi-mate a mixed model design in terms of optimal sensitivity and level ofinference without increasing false positive rates (McIntosh andLobaugh, 2004; Strother et al., 2002). The exact number of times thepermuted singular values exceeded the observed singular value wascomputed and pb .05 was considered significant. The numericalweights of the voxels comprising the brain LV are called “saliences”and can be positive or negative, indicating themagnitude and directionin which each voxel covaries with the corresponding design contrast.

The reliability of the brain regions comprising a LV was deter-mined via bootstrap estimation. Specifically, the standard error foreach voxel salience was calculated from a distribution of saliencesderived from resampling subjects 100 times with replacement and

recalculating the “nonrotated” task PLS on each sample. The ratio ofthe observed salience to the bootstrap standard error, which isapproximately equivalent to a z score, was then calculated to deter-mine reliability. Voxels were considered reliable if the bootstrap ratio(BSR)N |±3.62| (pb .0001).

The regions comprising a LVare reported in terms of clusters ofvoxels. A cluster within the LV was defined as at least 20 reliablecontiguous voxels and represented by its peak voxel, defined as thevoxel with the highest BSR.Where a cluster comprised several brainregions, the local maximum within each brain region was identifiedand reported. Regions were characterized in terms of anatomicalregion and Brodmann area (BA). The main findings are summarizedand the detailed reports are provided as supplemental material.

Seed PLSSeed PLS was applied to identify distributed patterns of activity

that were functionally connected (correlated) with a specified brainregion (“seed”) and compare these patterns between group andconditions. Seed PLS is identical to a “rotated” task PLS except thatthe columns of the design matrix comprise normalized rCBF activityfrom one or more voxels representing the activity of brain regions ofinterest during each condition. This matrix is then correlated with thedata matrix to produce a “cross-block correlation” matrix. SVD isperformed to extract commonalities and differences in this correlationmatrix (McIntosh, 1999) and permutation testing provides an assess-ment of significance. For the seed PLS, voxels comprising the LVswere considered reliable if the BSR exceeded |±2.33| (pb .01). Theexperimental effects are depicted graphically by plotting thecorrelation of the seed voxel(s) activity with the latent variable scorewithin group and condition. The salience associated with each regionis interpreted in terms of positive and negative correlations with theseed. When group differences are observed using a seed PLS, thevoxel salience of regions in the cluster report and projection plotrepresents group differences in correlations. Criteria for a voxel to bechosen as a seed included: 1) functional relevance supported by priorIBS research, 2) functional relevance to the hypothesized networks,and 3) significant and reliable functional connectivity empiricallysupported by the task PLS. Based upon these criteria, bilateralamygdala and right thalamus were selected as seed voxels.

Effective connectivity

Seed PLS identifies a pattern of brain regions functionally relatedto regions of interest. However, the correlation of two areas with athird does not reflect mutual correlation of the areas (Stephan, 2004).Instead, path analysis using a structural equation modeling frame-work (McIntosh and Gonzalez-Lima, 1991, 1994; McIntosh et al.,1994) was applied to simultaneously quantify the interactionsamong brain regions and to test for sex differences in the effectiveconnectivity of a hypothesized network.

Effective connectivity analyses require a priori specification of astructural (anatomical) model for testing. The structural modelrepresents the hypotheses about the causal relations between brainregions. Guided by the principle of parsimony, the nodes of thenetwork to be characterized and tested were selected based upon:1) a fundamental role in the networks hypothesized to be operatingduring response to an aversive pelvic visceral stimulus (i.e.,“homeostatic-afferent”, “emotional-arousal”, “cortical-modulatory”),2) a reliable loading on the sex-related network of regions revealedby the seed PLS, and 3) consistency with functional neuroimagingstudies providing information on the neural circuitry of pain

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(Petrovic and Ingvar, 2002; Ploghaus et al., 2003, 2001; Porro,2003; Porro et al., 2003; Price, 2000) and emotion (Pezawas et al.,2005; Stein et al., 2007). The causal structure among the brainregions comprising the network was supported by neuroanatomicalstudies (Amodio and Frith, 2006; Augustine, 1996; Cavada et al.,2000; Craig, 2002b, 2003a,c; Kringelbach, 2005; LeDoux, 2000;Ongur and Price, 2000; Price, 2003, 2005; Van Hoesen, 1995; Vogt,2005; Vogt et al., 2005). Although reciprocal top-down (descend-ing) and bottom-up (ascending) connectivity between regions iswell-known, there are mathematical restrictions on the number ofreciprocal pathways that can be specified for a given model usingstructural equation modeling (SEM) (Berry, 1984). Therefore, wemodeled most paths between structures as unidirectional, empha-sizing top-down connectivity between regions relevant for testinghypotheses regarding cortical modulation of the “homeostatic-afferent” and “emotional-arousal” networks, and bottom-up con-nectivity to test the hypotheses regarding ascending visceral input.

Having established a structural model, path analysis via a SEMframework was performed using Amos 6.0 (Arbuckle, 2005). Resid-ual variances, representing external input into the system (e.g.,unspecified regions, psychological characteristics, hormonal milieu),were fixed at 35% (McIntosh and Gonzalez-Lima, 1994) of theobserved regional variances within group and condition. SEM is amultivariate covariance-based analysis that determines the pathcoefficients or coupling between brain regions in a specified networkmodel. Expressed in terms of neural pathways, a path coefficient is thedirect proportional influence that one brain region has on another via adirect anatomical connection, controlling for all other regions in themodel. Path or beta coefficients reflect how a one unit change in onearea influences activity in the region to which it projects. NormalizedrCBF activity from each scan was extracted from the most reliablevoxel of each region selected as a node of the network and entered intoa path analysis.

Sex differences in the effective connectivity of the networkwere tested using multi-group tests for invariance (Joreskog, 1971).Specifically, sex differences in the circuitry of the network werelocalized using pair-wise comparisons between a completelyunconstrained model and a partially constrained model using achi-square difference test with 1 degree of freedom. Sequentially,each path of interest was restricted to be equal across sex and testedagainst a completely unconstrained model (e.g., all parametersestimated freely). Significance indicates that a pathway should befreely estimated in a model rather than constrained to be equal anddenotes significant differences between males and females in theeffective connectivity of the brain regions. These differences caninvolve both the sign and magnitude of the coefficient. Differences insign reflect a reversal or qualitative change in regional interactions.Changes inmagnitude reflect increase or decrease in the strength of thecoupling between regions. Chi-square statistics for group differencesand critical ratios for path coefficients were interpreted as significant atpb .05.

Results

Level 1 analysis: task PLS

Task PLS was employed to identify distributed patterns ofregions that relate to the brain's response to aversive visceralstimulus and its expectation. Significant LVs were found for bothmain effect contrasts, but not the interaction effect contrastsinvolving sex.

BL versus INF

Common INF-related network. The first significant LV (LV1)from the task PLS represented a pattern of brain regions thatmaximally distinguished between the BL and INF scans for bothfemales and males. This LV explained approximately 35% of thevariance in the “cross-block covariance” matrix. Permutation testingindicated significance at pb .0001. Reliable increases in brain activitywere evident during the response to an aversive visceral stimulus in anetwork of brain regions that included known “homeostatic-afferent”(thalamus, anterior INS (aINS), dACC, medial OFC (mOFC)), andPFC and parietal (BA 40) “cortical-modulatory” regions (see Supple-mental Table 1). As expected, the thalamus and INS contributedsignificantly to the discrimination of BL and INF. The thalamus wasselected as a seed voxel to test for sex differences in the functionalconnectivity of a “homeostatic-afferent” network.

BL versus EXP

Common EXP-related network. The second significant LV (LV2)from the task PLS characterized a functional pattern of regions thatmaximally differentiated BL from EXP for both females and males.LV2 accounted for 28% of the variance in the “cross-blockcovariance” matrix and permutation testing indicated significanceat pb .0001. Greater activity was observed during EXP in limbic andparalimbic regions (bilateral amygdala, parahippocampal regions,infragenual cingulate (iACC) (BA 25) and posterior cingulate cortex(PCC) (BA 30)), posterior dorsal INS (pdINS), dorsal prefrontalcortex (dPFC) (BA 10), right parietal (BA 40) regions and visualcortex (see Supplemental Table 2). Deactivations during EXP wereobserved in the right mPFC and the left OFC. As expected strongbilateral amygdala activation was evident and considered the mostappropriate seed to test for sex differences in the functional con-nectivity of an “emotional-arousal” network.

Level 2 analyses: seed PLS

Seed PLS was used to identify networks operating in concert withthe thalamus and amygdala regions identified in the task PLS and todetermine the influence of condition and sex on these networks.

Common thalamo-centric network. Seed PLS with the rightthalamus region (MNI −4,−8, 4), identified by the task PLS as beingreliably activated during INF as compared to BL, revealed a networkof regions correlating with the right thalamus and operating similarlyfor men and women across all conditions (see Supplemental Fig. 1).The projection plot in Fig. 1 depicts this common thalamo-centricnetwork of regions which explained 43% of the variance in the rightthalamus-whole-brain activity “cross-correlation” matrix. Permuta-tion testing indicated significance at pb .001. The network included“homeostatic-afferent” regions (bilateral thalamus, dACC/mid-cingulate cortex (MCC) (BA 24)), and bilateral INS (left aINS,right pdINS) as well as sensory (BA 1/40), motor (BA 6, 3),midbrain (ventral tegmental area), and prefrontal pain and affectsmodulating regions (PFC, OFC) (BA 44, 45, 47) (for details, seeSupplemental Table 3). In general, “homeostatic-afferent” regions(left thalamus, aINS, MCC), midbrain, prefrontal and parietalcortical regions (BA 40, BA 44, 45, 47) and sensory (BA 1) regionsdemonstrated positive correlations with the right thalamus whereasmotor, premotor areas (BA 4, 6) demonstrated negative correlations(for details, see Supplemental Table 3). To further test the hypothesisregarding sex differences in the “homeostatic-afferent” network, we

Fig. 1. Projection plot of the common thalamo-centric network. The plot depicts the network of brain regions that reliably correlated with the right thalamus (MNI4, 8, 4) across conditions and sex. Blue regions indicate regions with negative salience and correlated negatively with the thalamus. Red regions denote areas ofpositive salience that correlated positively with the thalamus. Sagittal section shows location of selected axial slices in the Z-plane of MNI space.

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performed an additional seed PLS using the aINS identified in thetask PLS but again results did not reveal a significant sex-relatednetwork (not shown).

Amygdalo-centric networksCommon amygdalo-centric network. Based on the a priori

hypothesis regarding the role of the amygdala and a related“emotional-arousal” network in central pain modulation, a seedPLS was performed with the amygdalae (MNI ±20,−5,−17)identified as more activated during EXP as compared to BL in thetask PLS. PLS seeded with the amygdala bilaterally revealed twosignificant amygdalo-centric networks. The first LV (LV1) revealeda network of regions correlated with the amygdalae that operatedsimilarly for females and males across all conditions (with thepossible exception of the left amygdala during baseline for femalessince the 95% confidence interval of the average correlation includeszero) (see Supplemental Fig. 2). The projection plot in Fig. 2 depictsthe common amygdalo-centric network. This network accounted for

36% of the variance in the amygdalae-whole-brain activity “cross-correlation” matrix (pb .001). This network of regions includedknown regions of the “homeostatic-afferent” network (left thalamus,pons, bilateral ventral pINS), limbic and paralimbic (hippocampus,MCC) and OFC and PFC regions (BA 9, 10, 46, 11/47) (for details,see Supplemental Table 4). In general, across conditions and sex,OFC, dlPFC, dPFC, mPFC, andMCC correlated negatively with thebilateral amygdala activity, consistent with known cortico-limbicinhibitory influences. In contrast, the bilateral mOFC, INS, and ponsdemonstrated positive correlations with the amygdalae.

Sex-specific amygdalo-centric network (LV2). The second LV(LV2) from the bilateral amygdala seed PLS revealed a network ofregions that demonstrated a consistent relationship with theamygdale across conditions (again with the possible exception ofthe left amygdala for females during BL) but operated differentlyfor females and males (see Supplemental Fig. 3). That is, thisnetwork of regions showed strong sex differences in functionalconnectivity with the amygdalae. The projection plot in Fig. 3

Fig. 2. Projection plot of the common amygdalo-centric network. The plot shows the network of brain regions that correlated with the right and left amygdalae(MNI ±20,−5,−17) similarly across conditions and sex. Red denotes regions of positive salience that correlated positively with the amygdalae whereas bluedenotes regions of negative salience that correlated negatively with the bilateral amygdala activity. Sagittal section shows location of selected axial slices in theZ-plane of MNI space.

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depicts this sex-related amygdalo-centric network that accountedfor 16% of the variance in the bilateral amygdala-whole-brainactivity “cross-correlation” matrix. Permutation testing indicatedsignificance at pb .01. The regions comprising this networkincluded “homeostatic-afferent” (bilateral thalamus, bilateralpINS), somatosensory (BA 40/7, BA 3/2/1, PCC), prefrontal(mPFC/mOFC (BA 10/11/47)), iACC and supragenual ACC(sACC) and pontine (PAG and LCC) regions. On average andacross conditions, the mOFC, iACC, sACC, and PAG/LCCdemonstrated more positive correlations with the amygdala forfemales but were characterized by either less positive or morenegative correlations in males (see Supplemental Table 5).Similarly, “homeostatic-afferent” regions (thalamus, pINS) andsomatosensory regions were more positively correlated with theamygdala for men, but showed either less positive or morenegative correlations with the amygdala bilaterally for women.Table 1 shows a summary of the various networks identified by thetask and seed PLS.

Effective connectivity

As described in the methods, brain regions comprising thenetwork to be tested were selected from the sex-related amygdalo-centric network (LV2). The connectivity between the nodes of theregions was specified to permit assessment of sex differences in theeffective connectivity of networks hypothesized to be involved inresponse to aversive pelvic stimuli. As can be seen in Fig. 4 andTable 2, the network model to be tested comprised “emotional-arousal” (amygdala→ iACC→sACC→amygdala→pons/LCC→amygdala, pons/LCC→sACC, pons/LCC→ iACC), “homeostatic-afferent” (thalamus→pINS, thalamus→sACC, thalamus→mOFC,pINS→mOFC) and “cortical-modulatory” (mOFC→ iACC,mOFC→sACC, mOFC→amygdala, iACC→pINS, pINS→amyg-dala) circuitries.

“Emotional-arousal” network. As shown in Fig. 5 and Table 2, ingeneral during all 3 conditions (BL, EXP, and INF), the female subjectsshowed stronger coupling between nodes of the “emotional-arousal”

Fig. 3. Projection plot of sex-related amygdalo-centric network. The plot shows the network of regions that reliably correlating with amygdalae (MNI ±20,−5,−17)across conditions. For females, blue regions indicate regions correlated negatively with the bilateral amygdala activity and yellow regions denote areas that correlatedpositively with the amygdalae. For men, blue regions indicate regions correlated positively with the amygdalae and yellow regions denote areas that correlatednegatively. Sagittal section shows location of selected axial slices in the Z-plane of MNI space.

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network. Themajority of the significant sex differences in this circuitrywere evident during EXP (Fig. 5, right panel, Table 2). During EXP,males and females differentially engaged the amygdala-iACC-sACC-

Table 1Summary of the networks identified by the task and seed PLS

Network PLS Designmatrix

LV Varianceexplained

p Sexdifference

INF-related Task INF-BL 1 35% b .0001 NoEXP-related EXP-BL 2 28% b .0001 NoCommon thalamo-

centricSeed Thalamus 1 43% b .001 No

Commonamygdalo-centric

Seed Bamygdala

1 36% b .001 No

Sex-relatedamygdalo-centric

2 16% b .01 Yes

Abbreviations: BL— baseline, INF— inflation, EXP— expectation, LV—latent variable, P — probability, PLS — partial least squares.

pons/LCC circuitry resulting in significant sex differences. Further-more, the amygdala→ iACC and amygdala→pons circuits showedconsistent sex differences across all conditions with stronger positivecoupling for females and negative or nonsignificant connectivity inmales.

Homeostatic-afferent network. As shown in Fig. 5 andTable 2, therewere only a small number of significantly activated circuits among theascending input from the thalamus and pINS projections to sACC andmOFC in either female or male subjects, reflecting only minor groupdifferences in effective connectivity. INS connectivity to mOFC wasconsistently negative during all 3 conditions in males and morepositive in women, but a statistically significant group difference wasonly found during the EXP condition (Fig. 5, Table 2). The connec-tivity between the thalamus and mOFC was more negative in femalescompared to men but no significant group differences were observed.

Cortical-modulatory network. There were only a few signifi-cant group differences in cortical–subcortical-modulatory circuits(see Fig. 5, Table 2).Whilemales consistently showed greater positiveconnectivity between the pINS→amygdala, with the strongest con-nections during BL and EXP, females showed weaker connectivity or

Fig. 4. Network nodes and schematic neural circuitry underlying responses to expectation and experience of aversive visceral stimulus. The structural model forthe proposed network to be tested was comprised of “emotional-arousal” (orange), “homeostatic-afferent” processing (blue) and “cortical-modulatory” circuitries(green). The table inset on the right gives MNI coordinates for network nodes shown in the left. Abbreviations: BA — Brodmann areas, Amyg — amygdala,iACC— infragenual cingulate cortex, INS— insula, LCC— locus coeruleus complex, mOFC— medial orbital frontal cortex, sACC— supragenual anteriorcingulate cortex, Thal — thalamus, ROI — region of interest.

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lack of engagement of this circuit. These group differences werestatistically significant for the BL and EXP conditions. In addition,females showed a strong positive connectivity between mOFC→amygdala during the INF and EXP conditions whereas males demon-strated weak negative connectivity in this circuitry, resulting in asignificant group difference.

Discussion

The primary goal of the current study was to identify brainnetworks activated by expected and delivered aversive pelvic vis-ceral stimuli in male and female patients with chronic abdominal

Table 2Effective connectivity parameter estimates and between-group comparisons of hom

Circuitry Connectivity Baseline

Fem Male

Beta Beta χ2Δ

Homeostatic-afferent Thal→ sACC 0.15 −0.28 1.5Thal→ INS 0.13 0.08 0.1Thal→mOFC −0.17 0.43 1.7INS→mOFC −0.21 −0.66 0.4

Emotional-arousal sACC→Amyg −0.01 −0.29⁎ 1.6iACC→ sACC 0.02 0.43⁎⁎ 1.8Amyg→ iACC 0.34⁎⁎ −0.16⁎ 13.2Amyg→Pons 0.48 0.16 0.6Pons→Amyg 0.05 0.08 0.1Pons→ iACC −0.21⁎⁎ −0.02 9.9Pons→ sACC −0.09 0.10 1.9Pons→mOFC 0.54⁎⁎ 0.12 4.8

Cortical-Modulatory mOFC→ iACC 0.08 0.11 0.1iACC→ INS −0.21⁎ −0.06 0.8INS→Amyg −0.20 0.60 4.5mOFC→Amyg 0.08 0.25 0.8

Critical values for the chi-square difference test (Δχ2) are 3.84, pb05, 6.64, pb .01,statistics. Significant beta coefficients are denoted by *pb .05 and **pb .01. Abbreviinfragenual cingulate cortex, INS— insula, LCC— locus coeruleus complex, mOcortex, Thal — thalamus.

pain, and to test for sex differences in the hypothesized circuitrywithin these networks. Multivariate network analyses using partialleast squares and structural equation modeling provided support forthe involvement of regions comprising “homeostatic-afferent”,“emotional-arousal” and “cortical-modulatory” networks. Whilethe brain of male and female patients showed a great degree ofsimilarity in its response to the experimental stimuli, significantsex-related differences in the functional and effective connectiv-ity of brain regions were demonstrated. To our knowledge, thisis the first demonstration of such sex-related differences in theeffective connectivity of brain networks in an experimental painparadigm.

eostatic-afferent, emotional-arousal, and cortical-modulatory circuits

Inflation Expectation

Fem Male Fem Male

Beta Beta χ2Δ Beta Beta χ2Δ

−0.08 −0.21 0.1 −0.57 0.14 3.4−0.09 0.11 1.7 0.20 0.01 0.7−0.64 −0.15 1.8 −0.68 −0.03 2.1−0.08 −0.32⁎⁎ 3.8 0.25⁎⁎ −0.08 9.70.07 −0.11 1.0 0.54⁎⁎ −0.44⁎ 17.4

−0.08 0.60⁎⁎ 4.4 −0.16 0.59⁎⁎ 7.10.28 −0.01 1.7 0.32⁎⁎ −0.19⁎ 12.70.75⁎ 0.36 0.5 0.96⁎⁎ −0.05 7.3

−0.07 0.08 2.0 0.13⁎ 0.11 0.00.05 −0.03 0.8 −0.25⁎⁎ 0.02 9.5

−0.13 0.16 3.5 −0.30⁎ 0.26⁎⁎ 11.9−0.58 0.21 3.6 −0.81 0.23 5.10.27⁎⁎ 0.06 2.8 0.10 −0.06 1.00.03 −0.26⁎ 3.4 0.07 −0.07 0.50.03 0.26 1.2 0.08 0.93⁎⁎ 9.00.58⁎⁎ −0.16 21.1 0.52⁎ −0.20⁎ 16.6

and 10.8, pb .001. Bolded values indicate significant chi-squared differenceations: Amyg— amygdala, BA—Brodmann areas, Fem— female, iACC—FC— medial orbital frontal cortex, sACC— supragenual anterior cingulate

Fig. 5. Estimated effective connectivity of the proposed network comprising the “homeostatic-afferent”, “emotional-arousal”, and “cortical-modulatory circuits”.The operation of the proposed network (as estimated by the completely unconstrained model) during BL, INF and EXP (columns) is presented for females andmales (rows). The beta coefficients (effective connectivity) are depicted by the thickness and color of the arrows. Solid arrows represent a parameter estimate thatwas considered significantly different from zero whereas dashed lines represent nonsignificant coefficients. Red arrows represent positive coupling whereas bluearrows represent negative coupling. The legend depicts the magnitude of the coefficients associated with each thickness. Abbreviations: Amyg — amygdala,iACC— infragenual cingulate cortex, INS— insula, LCC— locus coeruleus complex, mOFC—medial orbital frontal cortex, n.s— nonsignificant, sACC—supragenual anterior cingulate cortex, Thal — thalamus.

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Brain networks activated similarly in males and females in responseto the delivery or expectation of an aversive visceral stimulus

Using task PLS, we confirmed previous findings of distinctactivations of brain regions during INF and EXP (reviewed inMayeret al., 2006). Delivery of the aversive stimulus was associated withgreater activity in regions closely connected to the thalamus, such asbilateral aINS and dACC as well as modulatory cortical regionsincluding the mOFC and PFC. In contrast, the EXP of the stimuluswithout actual delivery, was characterized by greater activation ofbrain regions closely connected to the amygdala (parahippocampalregions, iACC, pINS and OFC) some of which are part of a wellcharacterized “emotional-arousal network” (Pezawas et al., 2005;Stein et al., 2007). The parietal cortex (BA40) showed greateractivation during both EXP and INF conditions, consistent with thehypothesized role of this region in a distributed network, allocatingattentional resources to novel sensory information (Mesulam, 1998).Further supporting such a role of parietal cortex as part of anattentional network is our recent demonstration of decreased acti-vation of this posterior attentional network during both the deliveryand expectation of an aversive visceral stimulus as subjects become

more familiar with the experimental paradigm (Naliboff et al.,2006).

Common (e.g., sex-unrelated) thalamo-centric and amygdalo-centricnetworks activated during both delivery and expectation of anaversive visceral stimulus

Seed PLS demonstrated the activation of the respective brainregions within the context of thalamo-centric and amygdalo-centricnetworks operating similarly between men and women across thenonINF (BL, EXP) and INF conditions. The functional connectivity ofthe common thalamo-centric network generally agreed with knownneuroanatomicalmodels (Craig, 2002a, 2003b).On the other hand, thefunctional connectivity within the amygdalo-centric network gen-erally agreed with the postulated model of emotional regulation andarousal circuits (Pezawas et al., 2005; Stein et al., 2007). Thus, whileour findings confirm previous observations (using SPM and region ofinterest analyses) by our group and others on brain regions activatedduring aversive pelvic visceral distension (reviewed in Mayer et al.,2006), they formally demonstrate for the first time that these regionsare activated as part of distinct functional brain networks.

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Sex-related activation of a network during delivery and expectationof an aversive visceral stimulus

In addition to the common networks identified in the seed PLS,where connectivity to the seed region was unrelated to the patient'ssex, we identified a sex-related amygdalo-centric network operatingduring both the INF and nonINF (BL, EXP) conditions. The regionscomprising this network supported the involvement of “homeostatic-afferent”, “emotional-arousal” and “cortical-modulatory” circuitriesduring expected and delivered aversive visceral stimuli.

Effective connectivity modeling supported the hypothesis that themost consistent sex-related differences occur within an “emotional-arousal” network. For example, women showed consistently strongerconnectivity between the pons/LCC and the mOFC during allconditions, while males consistently showed much weaker engage-ment of this circuit. Even though the spatial resolution of the scannerlimits our ability to precisely localize the source of the pontineactivation to the LCC, ascending noradrenergic projections from theLCC to the (medial PFC/mOFC) would be a plausible neuroanato-mical correlate of this connectivity (Valentino et al., 1999). Similarly,while women showed consistently strong and positive connectivitybetween amygdala and pons/LCC and iACC, these connections wereall weaker and sometimes negative in men. The strongest and mostconsistent sex difference in the connectivity between these regionswas seen during the EXP condition (Fig. 5).

The connectivity within the major nodes of the “homeostatic-afferent” (thalamus, pINSand dACC) and “cortical-modulatory”networks showed only few sex-related differences.Men demonstratedconsistently negative coupling of ascending connections from thepINS to mOFC during all three conditions, while women showedmore variable connections (Fig. 5). However, significant groupdifferences in the input to the mOFC from the pINS were observedonly during the EXP condition.

Possible correlation of findingswith previously reported SPManalyses

Using SPM analysis of [15O] water PET data, we have previouslyreported sex-related differences in the brain's response to an aversivevisceral stimulus, despite overall similarity of activated regions(Berman et al., 2000, 2006; Naliboff et al., 2003). When comparingmale and female IBS patients, greater mid-pINS and aINS activationwas found during INF in male patients in two different samples(Berman et al., 2000; Naliboff et al., 2003) and greater activation ofmOFC, dACC and amygdala in female patients in one study (Naliboffet al., 2003). The greater aINS activation in men was replicated in athird sample of healthy men and women using fMRI (Berman et al.,2006). In the current analysis, significant sex differences were ob-served in the connectivity of the pINS with the amygdala (during BLand EXP), and of the pINS with the mOFC (during EXP). Thesegroup differences were related to men showing a greater positiveconnectivity between the pINS and amygdala during the nonINFconditions, and a lack of coupling between the pINS and mOFCduring EXP. When viewed together, one may speculate that in maleIBS patients, the consistently greater activation of the INS during INFand sex-related differences in amygdala connectivity seen in thecurrent analysis may underlie the greater sympathetic nervous systemresponses previously reported in male IBS patients (Tillisch et al.,2005). Future studies with simultaneous recording of sympatheticnervous system activity are needed to confirm this hypothesis. On theother hand, the greater activation of the amygdala seen in the previousSPM analysis in women may be related to the greater positive

connectivity that the amygdala receives fromOFC and to the absenceof the feedback inhibition from sACC to the amygdala during the EXPcondition (Fig. 5).

Possible correlation of findings with sex differences in behavioralresponses

Using validated psychophysiological techniques, we have pre-viously reported that women with IBS show greater subjectiveemotional and perceptual responses to colorectal distension thanmalepatients or healthy men or women (Chang et al., 2006a). One mayspeculate that the sex-related differences in the connectivity of the“emotional-arousal” network seen in the current study may be relatedto these greater emotional and perceptual responses previouslyreported. However, in the current study, ratings were only obtained10min after each stimulus (BL, EXP, INF), and these limited post hocperceptual ratings did not reveal any sex differences (Naliboff et al.,2003). Although the possibility of sex differences in ascendingvisceral afferents may exist (Verne and Price, 2002), PLS seeded withknown ascending visceral pain regions (thalamus, anterior INS)indicated common functional connectivity between men and women.Sex differences in thalamus connectivity were only revealed by takingthe amygdala influence into account. Thus, our findings are mostconsistent with the hypothesis that sex-related differences are pri-marily a consequence of differences in the engagement of “emotional-arousal” networks and to a lesser degree, “cortical-modulatory” inputto this network, and that ascending information from homeostaticafferents is not driving the observed sex differences.

Strengths and limitations of connectivity modelingof brain responses

Path analysis via an SEM framework was used to test for sexdifferences in hypothesized circuitry, rather than to derive the best-fitting network model. We consider this approach reasonable giventhe small sample size and the limited number of data points availablein our data. Although determining the best-fitting model (i.e., causalstructure of the network) is highly desirable, inferences regardinggroup differences in effective connectivity have been shown to bevalid regardless of themodel fit (Protzner andMcIntosh, 2006). Alsoof note, the current effective connectivity analysis did not allowfor the precise estimation of network functioning during each con-dition due to biased parameter estimation in large networks withsmall samples. Greater precision of estimates can be obtained bysacrificing the completeness of a model (reducing the number ofnodes and connections) or with greater sample sizes. In addition, theproposed model is incomplete since the external input into thecurrent model (represented in the error terms, contextual factors suchas psychological and hormonal milieu) remains to be incorporated.Also, many of the regions comprising the network have reciprocalconnections with each other but could not be modeled due tomathematical constraints (Berry, 1984). Arguably other key brainregions and circuitry may remain to be delineated as well.Constructing and validating the most parsimonious model ofeffective connectivity during experience of aversive visceral stimuliwere beyond the scope of this paper.

Although the current approach enabled testing of importanthypotheses regarding the sex differences in effective connectivity ofneural networks in IBS patients, connectivity analyses with PETdata have several limitations, including its limited temporal andspatial resolutions. For example, the spatial resolution of PET

1042 J.S. Labus et al. / NeuroImage 41 (2008) 1032–1043

precludes localization of small brainstem nuclei and we can onlyspeculate on the involvement of such regions as the LCC. Also, as inthe current study, brain responses acquired with [15O] water PET areusually averaged over a 60–120 second period to achieve anacceptable signal to noise ratio (SNR). Such averaging results in theconvolution of multiple neural processes, presumably occurring atdifferent time scales (i.e., homeostatic-afferent processing, emotional-arousal, cortical modulation) in the overall picture of neural ac-tivation, and ignores the temporal engagement of this circuitry. Forexample temporal resolution of the postulated sequential processingof visceral afferent information within different INS subregionscannot be achieved reliably in the current data set. Network analyseswith data obtained with fMRI, MEG and EEG would be prudent asthis methodology can more realistically capture the temporaldynamics of the proposed circuitry.

Conclusions

In summary, the results of formal network analyses suggest theactivation of distinct yet overlapping networks concerned with theprocessing of ascending visceral information, emotional-arousal(induced by expectation or actual experience of the stimulus) andmodulatory cortical influences. While there was common activa-tion of these networks in both sexes, network functioning duringexpectation was uniquely characterized by sex differences in thecortico-limbic circuits involved in emotional-arousal, pain facil-itation and autonomic responses. These sex differences in specificbrain circuits may have implications for a better understanding ofIBS pathophysiology and for the reported sex differences in theeffectiveness of certain pharmacologic treatments. Our findingsalso suggest, that despite multiple peripheral and spinal mechan-isms proposed for sex differences in pain sensitivity, the peripheralsignal from the gut in IBS patients appears to contribute little tooverall sex differences at the level of the brain. Further workwill be necessary to confirm the functional relevance of thesenetworks via correlation with exogenous measures of attention,arousal, vigilance, emotional factors, and autonomic functioning.Despite these limitations, effective connectivity analyses per-mitted the first assessment of how brain regions interact with oneanother in the context of larger neural networks in patients withIBS.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.neuroimage.2008.03.009.

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