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Central autonomic network alterations in anorexia nervosa following peripheral adrenergic stimulation

Open AccessPublished:December 21, 2022DOI:https://doi.org/10.1016/j.bpsc.2022.12.009

      Abstract

      Background

      Anorexia nervosa (AN) is characterized by low body weight, disturbed eating, body image disturbance, anxiety, and interoceptive dysfunction. However, the neural processes underlying these dysfunctions in AN are unclear. This investigation combined an interoceptive pharmacological probe, the peripheral beta-adrenergic agonist isoproterenol, with resting state functional magnetic resonance imaging (rsfMRI) to examine whether individuals with AN relative to healthy comparisons (HC) show dysregulated neural coupling in central autonomic network brain regions.

      Method

      23 weight-restored AN female and 23 age, and BMI-matched HCs completed rsfMRI scanning before and after receiving isoproterenol infusions. We examined whole-brain functional connectivity (FC) changes using central autonomic network seeds in the amygdala, anterior insular cortex, posterior cingulate cortex, and ventromedial prefrontal cortex after performing physiological noise correction procedures.

      Results

      Relative to HCs, adrenergic stimulation caused widespread FC reductions in the AN group between central autonomic network regions and motor, premotor, frontal, parietal, and visual brain regions. Across both groups, these FC changes were inversely associated with trait anxiety (STAI-trait), trait depression (PHQ-9), and negative body image perception (Body Shape Questionnaire) measures but not with changes in resting heart rate. These results were not accounted for by baseline group FC differences.

      Conclusions

      Weight-restored AN females show a widespread state-dependent disruption of signaling between central autonomic, frontoparietal, and sensorimotor brain networks that facilitate interoceptive representation and visceromotor regulation. Additionally, trait associations between central autonomic network regions and these other brain networks suggest that dysfunctional processing of interoceptive signaling may contribute to affective and body image disturbance in AN.

      Trial Registration

      ClinicalTrials.gov Identifier: NCT02615119

      KEYWORDS

      Introduction

      Anorexia nervosa (AN) is an eating disorder defined clinically by the subjective disturbance of one’s own body image, and preoccupation with reducing one’s own body size, despite a low objective body mass (

      American Psychiatric Association (2022): Diagnostic and statistical manual of mental disorders (5th ed., text rev.). https://doi.org/10.1176/appi.books.9780890425787

      ). Beyond these consensus-based diagnostic features, it is characterized by the abnormal perception of homeostatic processes within the body including decreased hunger ratings or tolerance of sustained hunger sensations (
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      Neural Insensitivity to the Effects of Hunger in Women Remitted From Anorexia Nervosa.
      ,
      • Monteleone P.
      • Maj M.
      Dysfunctions of leptin, ghrelin, BDNF and endocannabinoids in eating disorders: beyond the homeostatic control of food intake.
      ), increased aversion to gastric fullness (
      • Perez M.E.
      • Coley B.
      • Crandall W.
      • Di Lorenzo C.
      • Bravender T.
      Effect of nutritional rehabilitation on gastric motility and somatization in adolescents with anorexia.
      ), decreased habituation to fullness sensations (
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      • Sunday S.R.
      Temporal patterns of hunger and fullness ratings and related cognitions in anorexia and bulimia.
      ,
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      • de Silva P.
      • Todd G.
      • Treasure J.
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      Thought–shape fusion in anorexia nervosa: an experimental investigation.
      ), and abnormal perception of cardiac and/or respiratory signals (
      • Khalsa S.S.
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      • Li W.
      • Vangala S.
      • Strober M.
      • Feusner J.D.
      Altered interoceptive awareness in anorexia nervosa: Effects of meal anticipation, consumption and bodily arousal.
      ,
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      Reduced perception of bodily signals in anorexia nervosa.
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      • Paulus M.P.
      • Bailer U.F.
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      Altered interoceptive activation before, during, and after aversive breathing load in women remitted from anorexia nervosa.
      ). These additional features suggest a broad dysfunction of interoception (
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      ,
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      ). However, few studies have examined how disrupted interoceptive processing in AN (
      • Khalsa S.S.
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      • Li W.
      • Vangala S.
      • Strober M.
      • Feusner J.D.
      Altered interoceptive awareness in anorexia nervosa: Effects of meal anticipation, consumption and bodily arousal.
      ,
      • Pollatos O.
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      Reduced perception of bodily signals in anorexia nervosa.
      ,
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      • Facchini F.
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      Temperament and character in eating disorders: ten years of studies.
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      • Pollatos O.
      • Herbert B.M.
      • Berberich G.
      • Zaudig M.
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      • Tsakiris M.
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      ,
      • Lapidus R.C.
      • Puhl M.
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      • Paulus M.P.
      • Rhudy J.L.
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      Heightened affective response to perturbation of respiratory but not pain signals in eating, mood, and anxiety disorders.
      ) might be linked to abnormal function of the central autonomic network (CAN) (
      • Benarroch E.E.
      The Central Autonomic Network: Functional Organization, Dysfunction, and Perspective.
      ), the primary brain network implicated in the processing of peripheral organ system (i.e., interoceptive) signals.
      Resting state functional magnetic resonance imaging (rsfMRI) studies have found that individuals with AN show evidence of disrupted functional connectivity (FC) in several brain networks, including the default mode and salience networks (
      • Gaudio S.
      • Wiemerslage L.
      • Brooks S.J.
      • Schioth H.B.
      A systematic review of resting-state functional-MRI studies in anorexia nervosa: Evidence for functional connectivity impairment in cognitive control and visuospatial and body-signal integration.
      ,
      • Steward T.
      • Menchon J.M.
      • Jimenez-Murcia S.
      • Soriano-Mas C.
      • Fernandez-Aranda F.
      Neural Network Alterations Across Eating Disorders: A Narrative Review of fMRI Studies.
      ), which contain regions crucial for interoception, such as the anterior cingulate and insular cortices (
      • Khalsa S.S.
      • Rudrauf D.
      • Feinstein J.S.
      • Tranel D.
      The pathways of interoceptive awareness.
      ,
      • Paulus M.P.
      • Khalsa S.S.
      When You Don’t Feel Right Inside: Homeostatic Dysregulation and the Mid-Insular Cortex in Psychiatric Disorders.
      ), and regions crucial for perception of the body’s exterior, including the precuneus and fusiform cortex (
      • Gaudio S.
      • Wiemerslage L.
      • Brooks S.J.
      • Schioth H.B.
      A systematic review of resting-state functional-MRI studies in anorexia nervosa: Evidence for functional connectivity impairment in cognitive control and visuospatial and body-signal integration.
      ,
      • Steward T.
      • Menchon J.M.
      • Jimenez-Murcia S.
      • Soriano-Mas C.
      • Fernandez-Aranda F.
      Neural Network Alterations Across Eating Disorders: A Narrative Review of fMRI Studies.
      ). From a neuroanatomical perspective the CAN overlaps with both the default mode (i.e. ventromedial prefrontal cortex (vmPFC) and posterior cingulate cortex (PCC)) and salience networks (i.e. amygdala and insula) (
      • Kleckner I.R.
      • Zhang J.
      • Touroutoglou A.
      • Chanes L.
      • Xia C.
      • Simmons W.K.
      • et al.
      Evidence for a large-scale brain system supporting allostasis and interoception in humans.
      ). However, its primary function is the hierarchical interoceptive processing of neurovisceral signals and the associated visceromotor and regulatory activity (
      • Smith R.
      • Thayer J.F.
      • Khalsa S.S.
      • Lane R.D.
      The hierarchical basis of neurovisceral integration.
      ). Accumulating task-based fMRI studies of AN also identify abnormal neural activity related to interoceptive processing. The insular and cingulate cortices have been particularly implicated in AN responses to, or anticipation of, aversive perturbations of interoceptive signals including nociceptive stimulation (
      • Strigo I.A.
      • Matthews S.C.
      • Simmons A.N.
      • Oberndorfer T.
      • Klabunde M.
      • Reinhardt L.E.
      • et al.
      Altered insula activation during pain anticipation in individuals recovered from anorexia nervosa: evidence of interoceptive dysregulation.
      ), gastric distension (
      • Perez M.E.
      • Coley B.
      • Crandall W.
      • Di Lorenzo C.
      • Bravender T.
      Effect of nutritional rehabilitation on gastric motility and somatization in adolescents with anorexia.
      ), and inspiratory breathing restriction (
      • Berner L.A.
      • Simmons A.N.
      • Wierenga C.E.
      • Bischoff-Grethe A.
      • Paulus M.P.
      • Bailer U.F.
      • et al.
      Altered interoceptive activation before, during, and after aversive breathing load in women remitted from anorexia nervosa.
      ). Abnormal activation of these regions in AN has also been observed relative to healthy comparisons (HC) during an interoceptive attention task performed under physiological resting conditions (
      • Kerr K.L.
      • Moseman S.E.
      • Avery J.A.
      • Bodurka J.
      • Zucker N.L.
      • Simmons W.K.
      Altered Insula Activity during Visceral Interoception in Weight-Restored Patients with Anorexia Nervosa.
      ). Collectively, these studies suggest that dysregulated neural monitoring of interoception in AN occurs across the continuum of homeostatic states.
      From a dynamic perspective, the CAN is proposed to exert “tonic, reflex, and integrative control of autonomic function [that] depends on the physiologic and behavioral state of the person” (
      • Benarroch E.E.
      The Central Autonomic Network: Functional Organization, Dysfunction, and Perspective.
      ). This implies the brain’s direct responsibility for monitoring, integrating, and ultimately, regulating interoceptive signaling. Such activities are likely facilitated by the coordinated synthesis of afferent autonomic information with not only homeostatic parameters, but also contextual information from somatic and behavioral/cognitive processes that are represented outside of CAN regions. Recently, FC within the CAN was found to be greater overall and more strongly correlated with heart rate in HCs compared to those with acute AN (

      Cruz Fdl, Schumann A, Suttkus S, Helbing N, Bar KJ (2022): Dynamic Changes in the Central Autonomic Network of Patients with Anorexia Nervosa. preprint.

      ). There is also some evidence of abnormal interactions among psychological traits and various cognitive functions in AN, including deficits in cognitive control, habit learning, value processing (e.g., reward learning), and heightened negative affect (e.g., trait anxiety, fear learning/generalization) (
      • Fassino S.
      • Amianto F.
      • Gramaglia C.
      • Facchini F.
      • Abbate Daga G.
      Temperament and character in eating disorders: ten years of studies.
      ,

      Friederich H-C, Herzog W (2010): Cognitive-Behavioral Flexibility in Anorexia Nervosa. Behavioral Neurobiology of Eating Disorders: Springer Berlin Heidelberg, pp 111-123.

      ,
      • Kaye W.H.
      • Wierenga C.E.
      • Knatz S.
      • Liang J.
      • Boutelle K.
      • Hill L.
      • et al.
      Temperament-based Treatment for Anorexia Nervosa.
      ,

      Steinglass JE, Berner LA, Attia E (2019): Cognitive Neuroscience of Eating Disorders. Psychiatric Clinics of North America. 42:75-91.

      ,
      • Zhang Z.
      • Robinson L.
      • Jia T.
      • Quinlan E.B.
      • Tay N.
      • Chu C.
      • et al.
      Development of Disordered Eating Behaviors and Comorbid Depressive Symptoms in Adolescence: Neural and Psychopathological Predictors.
      ). However, the links between these psychological traits and interoceptive processing are unclear.
      We addressed this gap in the current study by examining changes in CAN dynamics in AN following the pharmacologic perturbation of cardiorespiratory interoception. Specifically, we used rsfMRI to compare network-level changes in the blood oxygen-level-dependent (BOLD) signal induced by intravenous infusions of isoproterenol, an adrenaline analog with a short half-life that acts preferentially on peripheral beta-adrenergic receptors (
      • Borges A.C.R.
      • Feres T.
      • Vianna L.M.
      • Paiva T.B.
      Recovery of impaired K+ channels in mesenteric arteries from spontaneously hypertensive rats by prolonged treatment with cholecalciferol.
      ,
      • Murphy V.A.
      • Johanson C.E.
      Adrenergic-Induced Enhancement of Brain Barrier System Permeability to Small Nonelectrolytes: Choroid Plexus versus Cerebral Capillaries.
      ,
      • Olesen J.
      • Hougård K.
      • Hertz M.
      Isoproterenol and propranolol: ability to cross the blood-brain barrier and effects on cerebral circulation in man.
      ). We have consistently found isoproterenol to elicit focal changes in insular cortex activity using multiple fMRI sequence modalities in healthy participants (
      • Hassanpour M.S.
      • Yan L.
      • Wang D.J.
      • Lapidus R.C.
      • Arevian A.C.
      • Simmons W.K.
      • et al.
      How the heart speaks to the brain: neural activity during cardiorespiratory interoceptive stimulation.
      ,
      • Hassanpour M.S.
      • Simmons W.K.
      • Feinstein J.S.
      • Luo Q.
      • Lapidus R.C.
      • Bodurka J.
      • et al.
      The Insular Cortex Dynamically Maps Changes in Cardiorespiratory Interoception.
      ) and recently identified group differences in vmPFC activation in clinically anxious individuals (
      • Teed A.R.
      • Feinstein J.S.
      • Puhl M.
      • Lapidus R.C.
      • Upshaw V.
      • Kuplicki R.T.
      • et al.
      Association of Generalized Anxiety Disorder With Autonomic Hypersensitivity and Blunted Ventromedial Prefrontal Cortex Activity During Peripheral Adrenergic Stimulation.
      ). In behavioral studies, we have shown that AN individuals report heightened cardiorespiratory sensations during stimulation and during saline infusion (
      • Khalsa S.S.
      • Craske M.G.
      • Li W.
      • Vangala S.
      • Strober M.
      • Feusner J.D.
      Altered interoceptive awareness in anorexia nervosa: Effects of meal anticipation, consumption and bodily arousal.
      ), and abnormal bodily localization of interoceptive sensations (
      • Khalsa S.S.
      • Hassanpour M.S.
      • Strober M.
      • Craske M.G.
      • Arevian A.C.
      • Feusner J.D.
      Interoceptive Anxiety and Body Representation in Anorexia Nervosa.
      ). Thus, isoproterenol is a promising tool for illuminating the brain networks contributing to distorted interoceptive processing in AN.
      We evaluated CAN function in weight-restored AN females relative to female HCs matched on age and body mass index via rsfMRI scanning both before and following peripheral cardiorespiratory stimulation with isoproterenol. We provided participants a meal immediately following the experimental paradigm to enhance the relevance of affective and interoceptive processing during the scanning period, as prior studies have demonstrated that meal anticipation represents a clinically relevant context in which individuals with AN exhibit heightened meal-related anxiety (
      • Zhang Z.
      • Robinson L.
      • Jia T.
      • Quinlan E.B.
      • Tay N.
      • Chu C.
      • et al.
      Development of Disordered Eating Behaviors and Comorbid Depressive Symptoms in Adolescence: Neural and Psychopathological Predictors.
      ) and abnormal processing of isoproterenol-induced sensations (
      • Khalsa S.S.
      • Craske M.G.
      • Li W.
      • Vangala S.
      • Strober M.
      • Feusner J.D.
      Altered interoceptive awareness in anorexia nervosa: Effects of meal anticipation, consumption and bodily arousal.
      ,
      • Sheehan D.V.
      • Lecrubier Y.
      • Sheehan K.H.
      • Amorim P.
      • Janavs J.
      • Weiller E.
      • et al.
      The Mini-International Neuropsychiatric Interview (MINI): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10.
      ). Based on the hypothesis of abnormal CAN functioning within AN, we predicted group differences in pre- to post-infusion changes in whole-brain, voxelwise FC across several a priori CAN seed regions of interest (ROI). These ROIs consisted of the left and right anterior insula, the left and right amygdala, and the bilateral ventromedial prefrontal cortex (vmPFC) and posterior cingulate cortex (PCC). We also examined the dimensional relationship that traits relevant to AN, anxiety, depression, and body shape perception may have on changes in CAN FC following peripheral adrenergic stimulation.

      Methods

      Participants

      AN participants were required to meet lifetime DSM 5 criteria for a diagnosis of AN, and current diagnoses were verified using the clinician-administered Mini International Neuropsychiatric Interview (MINI version 6.0)(
      • Sheehan D.V.
      • Lecrubier Y.
      • Sheehan K.H.
      • Amorim P.
      • Janavs J.
      • Weiller E.
      • et al.
      The Mini-International Neuropsychiatric Interview (MINI): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10.
      ). However, all participants were required to be weight-restored as defined by a minimum body mass index (BMI) of 18.5 or greater for at least 1 month to control for the well-described state effects of acute starvation (
      • Wierenga C.E.
      • Bischoff-Grethe A.
      • Melrose A.J.
      • Irvine Z.
      • Torres L.
      • Bailer U.F.
      • et al.
      Hunger Does Not Motivate Reward in Women Remitted from Anorexia Nervosa.
      ). Self-reported race/ethnicity were also recorded.
      No participant had any lifelong history of neurological, diabetic, cardiovascular, or respiratory disorders, including asthma. Lifetime history of schizophrenia, bipolar disorder, or antisocial personality disorder were also exclusionary. However, to maintain a sample representative of community treatment samples, we allowed for comorbid mood and anxiety disorders except for panic disorder to reduce potential complications due to isoproterenol-induced panic anxiety. We also allowed participants taking selected psychotropic medications into the study provided the dose was stable during a 2-week period prior to scanning (see Supplemental Table S1). See supplement for pre-scan instructions regarding medications.
      This study was approved by the Western IRB. All participants provided written informed consent and were compensated for their participation. It was part of a larger project investigating interoceptive dysfunction AN and generalized anxiety disorder (
      • Teed A.R.
      • Feinstein J.S.
      • Puhl M.
      • Lapidus R.C.
      • Upshaw V.
      • Kuplicki R.T.
      • et al.
      Association of Generalized Anxiety Disorder With Autonomic Hypersensitivity and Blunted Ventromedial Prefrontal Cortex Activity During Peripheral Adrenergic Stimulation.
      )(ClinicalTrials.gov Identifier: NCT02615119). The project included not only the isoproterenol infusion perturbation task but also an interoceptive attention task, both performed between collection of the two rsfMRI scans reported here. On arrival, participants consumed a 300 Calorie snack. Immediately after the scan session all participants consumed a 1000 Calorie meal after choosing from one of three isocaloric options prior to the scanning session (as in (
      • Khalsa S.S.
      • Craske M.G.
      • Li W.
      • Vangala S.
      • Strober M.
      • Feusner J.D.
      Altered interoceptive awareness in anorexia nervosa: Effects of meal anticipation, consumption and bodily arousal.
      ,
      • Sheehan D.V.
      • Lecrubier Y.
      • Sheehan K.H.
      • Amorim P.
      • Janavs J.
      • Weiller E.
      • et al.
      The Mini-International Neuropsychiatric Interview (MINI): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10.
      ).

      Data acquisition

      Data were collected on a 3 Tesla General Electric (GE) MR750 MRI scanner with a GE 8-channel receive-only head coil. 32-channel EEG signals were recorded simultaneously but not analyzed as part of this study. First, high-resolution anatomical T1-weighted volume scans (MP-RAGE) with SENSE were obtained in sagittal orientation (TR=1900 ms, TE=2.26 ms, matrix 256×256, flip angle=9°, 186 slices, and an isotropic resolution of 1×1×1 mm3). Next, T2*-weighted images were obtained using a gradient-echo EPI sequence with Sensitivity Encoding (TR=2000 ms, TE=27 ms, acquisition matrix=128×128 mm2, flip angle=78°, 39 interleaved transverse slices of 2.9 mm thickness, and an in-plane resolution of 1.9×1.9 mm2). A total of 240 whole-brain volume sets were acquired in each 8-minute resting state session.

      Experimental design

      Two eight-minute eyes open resting state fMRI scans were collected, one before and the other following the completion an interoceptive perturbation and then an interoceptive attention task. During the interoceptive perturbation task, participants received six intravenous bolus infusions in a double-blinded, randomized manner: isoproterenol (0.5 micrograms (μg) and 2 μg) and saline, each administered twice. Isoproterenol was obtained from Valeant Pharmaceuticals. The isoproterenol task duration was 30 minutes. During the interoceptive attention task (adapted from (
      • Simmons W.K.
      • Avery J.A.
      • Barcalow J.C.
      • Bodurka J.
      • Drevets W.C.
      • Bellgowan P.
      Keeping the body in mind: insula functional organization and functional connectivity integrate interoceptive, exteroceptive, and emotional awareness.
      )) participants focused their attention on cardiac, respiratory, or stomach sensations, or on a visual target. No infusions were delivered during this 18-minute task. Thus, the resting state scans were collected approximately 50 minutes apart. During the resting state scans, cardiac and respiratory signals were recorded at 40 Hertz via MRI-scanner-equipped pulse oximeter attached to the nondominant index finger and a respiratory transducer belt placed around the torso. To assess key illness-related traits during the baseline visit, we administered several self-report scales including the Patient Health Questionnaire (PHQ-9; (
      • Kroenke K.
      • Spitzer R.L.
      • Williams J.B.
      The PHQ-9: validity of a brief depression severity measure.
      )) to assess depression symptoms, the state-trait anxiety inventory (STAI (

      Spielberger CD (1983): State-trait anxiety inventory for adults.

      )) to assess trait anxiety, and the body shape questionnaire (BSQ (
      • Cooper P.J.
      • Taylor M.J.
      • Cooper Z.
      • Fairbum C.G.
      The development and validation of the Body Shape Questionnaire.
      )) to assess perceptual and affective concerns about how the current body shape or appearance was experienced by persons with eating disorders (
      • Rosen J.C.
      • Jones A.
      • Ramirez E.
      • Waxman S.
      Body Shape Questionnaire: studies of validity and reliability.
      ). Vital signs (heart rate, systolic and diastolic blood pressure) were measured during the baseline visit and after meal completion at the end of the fMRI scanning visit.

      fMRI preprocessing

      Data analysis was performed using AFNI (
      • Cox R.W.
      AFNI: Software for Analysis and Visualization of Functional Magnetic Resonance Neuroimages.
      )⁠. After discarding the first five images to ensure a steady-state tissue magnetization, we identified volumes with excess motion (Euclidean norm of the motion derivatives>0.3 mm or fraction of voxel outliers>10%) and removed spikes in the signal intensity time courses. Slow BOLD fluctuations and time-locked cardiac and respiratory artifacts were removed using respiration volume per time (RVT) regressors (
      • Birn R.M.
      • Smith M.A.
      • Jones T.B.
      • Bandettini P.A.
      The respiration response function: the temporal dynamics of fMRI signal fluctuations related to changes in respiration.
      ) and the RETROICOR algorithm (
      • Glover G.H.
      • Li T.-Q.
      • Ress D.
      Image-based method for retrospective correction of physiological motion effects in fMRI: RETROICOR.
      )⁠. The RVT and RETRICOR regressors consisted of the RVT function and four delayed terms at 5, 10, 15, and 20 seconds and eight RETROICOR regressors generated on a slice-wise basis by AFNI's “RetroTS.m” script (
      • Jo H.J.
      • Saad Z.S.
      • Simmons W.K.
      • Milbury L.A.
      • Cox R.W.
      Mapping sources of correlation in resting state FMRI, with artifact detection and removal.
      )⁠. See Supplement for further preprocessing steps.

      Regions-of-interest and FC analysis

      To investigate functional abnormalities of autonomic brain regions, we defined six CAN regions of interest (ROI) implicated in cardiovascular regulation (
      • Benarroch E.E.
      The Central Autonomic Network: Functional Organization, Dysfunction, and Perspective.
      ,
      • Beissner F.
      • Meissner K.
      • Bar K.J.
      • Napadow V.
      The autonomic brain: an activation likelihood estimation meta-analysis for central processing of autonomic function.
      ) (Figure 1), namely the bilateral vmPFC, left and right amygdala, left and right anterior insular cortex (LAins, RAins), and bilateral PCC (
      • Thayer J.F.
      • Lane R.D.
      A model of neurovisceral integration in emotion regulation and dysregulation.
      ,
      • Thayer J.F.
      • Lane R.D.
      Claude Bernard and the heart–brain connection: Further elaboration of a model of neurovisceral integration.
      ). The vmPFC (MNI x=0, y=44, z=14) and PCC (MNI x=4, y=-56, z=22) ROIs were drawn as 10mm radius spheres (
      • Cooper P.J.
      • Taylor M.J.
      • Cooper Z.
      • Fairbum C.G.
      The development and validation of the Body Shape Questionnaire.
      ,
      • Rosen J.C.
      • Jones A.
      • Ramirez E.
      • Waxman S.
      Body Shape Questionnaire: studies of validity and reliability.
      )⁠, while the anterior insula and amygdala ROIs were created using the WFU Pick Atlas tool for SPM (
      • Maldjian J.A.
      • Laurienti P.J.
      • Kraft R.A.
      • Burdette J.H.
      An automated method for neuroanatomic and cytoarchitectonic atlas-based interrogation of fMRI data sets.
      ,
      • Maldjian J.A.
      • Laurienti P.J.
      • Burdette J.H.
      Precentral gyrus discrepancy in electronic versions of the Talairach atlas.
      ) to increase anatomical accuracy given the variable morphology and size of these structures. To define the anterior insula, we divided the whole insula ROI into two subregions using a perpendicular plane that cuts the posterior-anterior axis at y=6 and used the anterior section (
      • de la Cruz F.
      • Schumann A.
      • Kohler S.
      • Reichenbach J.R.
      • Wagner G.
      • Bar K.J.
      The relationship between heart rate and functional connectivity of brain regions involved in autonomic control.
      ).
      Figure thumbnail gr1
      Figure 1Location of central autonomic network (CAN) seed regions of interest: ventromedial prefrontal cortex (orange), posterior cingulate cortex (green), amygdala (left and right, red), and anterior insular cortex (left and right, blue).
      We obtained resting-state FC maps for each of the pre-infusion and post-infusion rsfMRI scans by extracting the time series from each seed region and correlated them against all brain voxels. The resulting correlation coefficients were then converted to Fisher z-statistics to produce a more normally distributed variable (

      Zar JH (1999): Biostatistical analysis. Pearson Education India.

      ). Next, we tested for group differences in the effects of isoproterenol on FC in healthy and AN individuals over time. All resulting clusters of this group x time interaction analysis were further corrected for multiple comparisons. For this, we used the 3dClustSim function in AFNI to run 10,000 Monte Carlo simulations of whole‐brain fMRI data and determine the cluster size at which the false positive probability was below α<0.05 with an uncorrected voxelwise threshold of p<0.001. For these thresholds, the simulation yielded a cluster size threshold of 130 contiguous voxels. Finally, we conducted an exploratory correlational analysis evaluating associations between FC changes and trait measures of anxiety (STAI-trait), depression (PHQ-9) and body image (BSQ), using the Bonferroni method for multiple comparison correction. This dimensional analysis was conducted across both groups as these traits are expected to vary across the spectrum of healthy and clinical conditions.

      Results

      Of the 70 individuals allocated to the intervention, 58 completed it with good quality data for both pre and post-isoproterenol rsfMRI scans (Figure S1). Six HCs were excluded for not completing all self-report measures. Another 6 HCs were removed by a propensity matching procedure (MatchIt in R (
      • Ho D.E.
      • Imai K.
      • King G.
      • Stuart E.A.
      Matching as Nonparametric Preprocessing for Reducing Model Dependence in Parametric Causal Inference.
      )), to obtain an equal analysis sample of 23 ANs and 23 HCs matched on age and body mass index (Table 1). The groups did not differ on blood pressure (BP) at baseline or resting heart rate (HR) during the pre-isoproterenol scan. However, the HC group showed evidence of increased systolic and diastolic BP relative to the AN group, whose values were unchanged. The AN group also showed higher HR during the baseline visit, the post-isoproterenol scan, and the end of the fMRI visit (Table 1).
      Table 1Descriptive and Inferential Statistics for Demographic and Diagnostic Variables
      HCANt-Stat (df)p-Value
      Mean (SD)Mean (SD)
      Gender2F23F
      Education (years)14.3(1.7)a13.7(1.3)t(38.93)=-1.21p=0.234
      Age (years)22.3(3.9)21.2(3.7)t(43.90)=-0.97p=0.338
      BMI22.9(2.8)22.4(3.2)t(43.23)=-0.61p=0.544
      Anxiety (STAI-Trait)28.6(7.4)53.1(10.1)t(40.24)=9.42p<0.001
      Depression (PHQ-9)0.8(1.2)7.6(5.4)t(24.12)=5.82p<0.001
      Body image (BSQ)46.7(10.9)127.0(36.6)t(25.89)=10.07p<0.001
      Physiological measures - Baseline
      Systolic BP (mmHg)113.3(10.0)110.2(10.3)t(43.98)=-1.03p=0.307
      Diastolic BP (mmHg)72.2(9.1)72.2(9.0)t(43.99)=-0.02p=0.987
      HR (bpm)69.2(9.1)76.9(12.5)t(40.20)=2.40p=0.021
      Physiological measures - During fMRI
      HR - pre ISO (bpm)65.5(11.4)69.8(7.4)t(37.67)=1.53p=0.135
      HR - post ISO (bpm)65.6(8.4)71.3(9.4)t(43.5)=2.34p=0.034
      Physiological measures - Post fMRI
      Systolic BP (mmHg)118.3(14.4)110.9(9.2)t(37.27)=-2.08p=0.045
      Diastolic BP (mmHg)78.0(8.2)73.0(7.3)t(43.30)=-2.19p=0.034
      HR (bpm)75.8(10.7)83.1(10.6)t(43.99)=2.34p=0.024
      a One healthy comparison participant did not provide their years of education.
      Abbreviations: AN, anorexia nervosa; BMI, Body Mass Index; BP, blood pressure; bpm, beats per minute; BSQ, Body Shape Questionnaire; ISO; isoproterenol; PHQ-9, Patient Health Questionnaire; F, Female; HC, healthy comparison; M, Male; STAI, State Trait Anxiety Index.
      Across all group x time interactions for each seed, the analysis of rsfMRI data revealed FC increases for the HC group and decreases for the AN group for a total of 19 significant multiple comparison corrected seed-to-target associations (Table 2). Specifically, FC change within CAN regions was observed between the vmPFC and both anterior insula regions, and the left anterior insula was coupled with the PCC. The right amygdala showed no significant FC change. Also prominently correlated with the CAN seeds were the sensorimotor and lateral prefrontal cortices. Figure 2 displays group x time (pre-post change) FC interaction maps for the left anterior insula, left amygdala, vmPFC, and PCC ROIs (see Supplement for group and condition effects).
      Table 2Brain regions showing Group x Time FC interaction.
      Seed and Target RegionsL/RBAVoxel CountPeak (MNI)T-Anxiety STAI-TraitBody Image BSQDepression PHQ-9
      XYZstatrprprp
      statstatstatstatstatstat
      Seed: Right Anterior Insula
      Target

      Medial PFC
      L/R104102.646.88.4-4.77-0.63p<0.001-0.58p<0.001-0.53p<0.001
      Primary MotorL4248-13.1-33.864.4-5.61-0.67p<0.001-0.58p<0.001-0.54p<0.001
      Seed: Left Anterior Insula
      Target

      Medial PFC
      L/R10440-0.967.8-3.9-4.54-0.56p<0.001-0.53p<0.001-0.48p<0.001
      Primary Sensory/MotorL1/4293-42.9-2966.1-5.45-0.55p<0.001-0.54p<0.001-0.39p=0.007
      PCC/PrecuneusL23/30/31260-14.9-56.520.6-4.26-0.54p<0.001-0.57p<0.001-0.41p=0.004
      Primary MotorL4204-14.9-30.264.4-5.15-0.65p<0.001-0.54p<0.001-0.51p<0.001
      Lingual/Mid-Occipital GyrusR1816027.1-84.5-3.9-4.46-0.60p<0.001-0.56p<0.001-0.43p=0.003
      Seed: Left Amygdala
      Target

      Posterior Insula/STG
      R13/2219044.6-14.56.6-5.20-0.62p<0.001-0.64p<0.001-0.52p<0.001
      Seed: Posterior Cingulate Cortex
      Target

      Parahippocampal/PCC/Cuneus
      L17/18/27/30462-20.1-75.88.4-4.85-0.53p<0.001-0.61p<0.001-0.44p=0.002
      MFGL10/46199-37.6383.1-4.36-0.57p<0.001-0.57p<0.001-0.48p<0.001
      IFGL11/47158-23.634.5-12.6-4.42-0.59p<0.001-0.60p<0.001-0.43p=0.003
      ParahippocampalR27/3015111.4-42.51.4-4.61-0.56p<0.001-0.64p<0.001-0.43p=0.003
      Seed: Ventromedial Prefrontal Cortex
      Target

      Superior Parietal/Supramarginal
      L7/40603-41.1-40.845.1-4.85-0.53p<0.001-0.50p<0.001-0.36p=0.015
      IFGR44/4522853.46.524.1-4.62-0.55p<0.001-0.57p<0.001-0.51p<0.001
      OFCL11/47187-25.432.8-10.9-4.86-0.65p<0.001-0.62p<0.001-0.47p<0.001
      IFG/PremotorL9/44174-53.41.222.4-4.39-0.51p<0.001-0.52p<0.001-0.33p=0.024
      OFCR11/4715827.132.8-9.1-4.70-0.58p<0.001-0.60p<0.001-0.44p=0.002
      LPFCL45/46158-41.1-34.510.1-4.60-0.57p<0.001-0.61p<0.001-0.21p=0.003
      PremotorL6151-27.1-5.864.4-4.36-0.57p<0.001-0.53p<0.001-0.43p=0.002
      The criterion for detecting significant clusters was p<0.05 as estimated by AFNI’s 3dClustSim (cluster size > 130 voxels, voxel level thresholded at p<0.001). Significant correlations with the trait facet of the State Trait Anxiety Inventory (STAI), Body Shape Questionnaire (BSQ), and Depression Patient Health Questionnaire (PHQ-9) were Bonferroni-corrected for multiple comparisons. Abbreviations: dlPFC, dorsolateral prefrontal cortex; FC, functional connectivity; IFG, inferior frontal cortex; PCC, posterior cingulate cortex.
      Figure thumbnail gr2
      Figure 2Top: T-maps of group x time interaction for the left anterior insula (blue), left amygdala (red), vmPFC (yellow) and PCC (green). Bottom: Interaction plots for selected clusters with connectivity values before (T1) and after isoproterenol infusion (T2), specifically for (A) the left anterior insula to mPFC, (B) the left amygdala to right insula, (C) the vmPFC to left parietal cortex. Abbreviations: AN, anorexia nervosa; HC, healthy comparison; mPFC, medial prefrontal cortex; PCC, posterior cingulate cortex; vmPFC, ventromedial prefrontal cortex.
      To evaluate associations between FC and traits relevant to AN, the change in FC for each of the 19 significant seed-to-target associations was then correlated with anxiety (indexed via STAI trait score), body shape perception (indexed via BSQ total score), and depression (indexed via PHQ-9 total score). We computed these associations across groups to explore whether they might suggest a transdiagnostic dimensional relationship. After applying Bonferroni correction for multiple comparisons (corresponding to a threshold of p<0.0026), all region pairs showed at least a moderate inverse correlation with the STAI and BSQ and similar but smaller magnitude correlation with the PHQ-9 (Table 2 and Figure 3). To ensure that the observed changes in FC following adrenergic stimulation were not simply reflecting pre-existing widespread group differences, we assessed for FC group differences in the baseline rsfMRI scans collected prior to the infusions. The only FC difference observed was a decrease for AN relative to HC between the PCC seed and the anterior PFC (MNI x=11, y=61, z=15) (Figure 4).
      Figure thumbnail gr3
      Figure 3Regional associations between changes in FC with trait measures of anxiety (STAI-Trait) and body image (BSQ) across AN and HC groups. For illustrative purposes, selected relationships are displayed: each scatterplot shown reflects the FC-trait association indicated by the black arrow in the adjacent glass brain. The black line in each plot indicates the correlation slope. Blue diamonds represent healthy individuals, and red circles represent AN individuals. All reported p-values are Bonferroni-corrected and two-tailed. Abbreviations: AIns, anterior insula; BSQ, Body Shape Questionnaire; IFG, inferior frontal gyrus; IP, inferior parietal; M1, primary motor cortex; PCC, posterior cingulate cortex; OFG, orbitofrontal gyrus; S1, primary somatosensory cortex; STAI: State Trait Anxiety Inventory; V1, primary visual cortex; vmPFC, ventromedial prefrontal cortex.
      Figure thumbnail gr4
      Figure 4FC differences between AN and HC for the PCC seed region at baseline (i.e., before receiving the isoproterenol infusion). A cluster of reduced functional connectivity was observed between the PCC seed and an anterior medial PFC cluster in AN. The cluster is significant at α  < 0.05 after cluster-based correction (> 130 contiguous voxels as determined by AFNI' 3dClustSim) with voxel-wise p < 0.001. For visualization purposes, the violin plot indicates the FC values of each group in the cluster. Abbreviations: FC, functional connectivity.
      Finally, to ensure that the observed changes in FC following adrenergic stimulation were not simply reflecting the observed post-scan group differences in BP and HR (Table 1), we evaluated and found no associations between CAN seed-to-target regions and the post-scan systolic and diastolic BP as well as HR (Supplement). We also found no significant association between changes in post-scan HR or BP and CAN seed regions for any cluster (Figure S3 and Supplement).

      Discussion

      In the current study, we observed strong evidence of changes in central autonomic network FC induced by a peripheral beta-adrenergic challenge in weight-restored individuals with AN relative to HCs. Specifically, FC was diminished between CAN seed regions and a wide range of brain areas in the AN group, as compared to HCs, who consistently showed opposing FC increases in those regions. The reductions in FC were observed both within the CAN and between the CAN and frontal, parietal, somatosensory, motor, and visual brain regions. Each correlated ROI pair also showed an inverse relationship with trait anxiety, trait depression, and a perceptual measure of body image disturbance, but showed no relationship with changes in resting heart rate or blood pressure, suggesting that interoceptive signaling between CAN regions and other brain networks may facilitate the representation and regulation of these affective and body image concerns.
      The widespread FC group differences occurred between regions of the CAN and portions of networks that primarily represent default mode/self-processing, cognitive control, motor, somatosensory, and visual processing. While we saw only one group difference in resting state FC prior to adrenergic stimulation (reduced PCC-to-mPFC connectivity in AN), our observed pre-post infusion FC changes align with frontoparietal and CAN FC differences seen previously in the cross-sectional rsfMRI AN literature (
      • Gaudio S.
      • Wiemerslage L.
      • Brooks S.J.
      • Schioth H.B.
      A systematic review of resting-state functional-MRI studies in anorexia nervosa: Evidence for functional connectivity impairment in cognitive control and visuospatial and body-signal integration.
      ). We observed several FC changes in the amygdala, but that was the only subcortical region for which we observed FC with another CAN region (i.e., the posterior insula), either as a seed or target. This is noteworthy as several CAN regions coordinate with subcortical regions in other circuits to integrate viscerosensory input and affective information to initiate visceromotor output (
      • Cechetto D.F.
      Central representation of visceral function.
      ,

      Loewy AD, Spyer KM (1990): Central Regulation of Autonomic Functions. Oxford University Press.

      ,

      LeDoux JE (1992): Emotion and the amygdala. The amygdala: Neurobiological aspects of emotion, memory, and mental dysfunction. New York, NY, US: Wiley-Liss, pp 339-351.

      ). Prior cross-sectional rsfMRI studies of AN have primarily observed altered FC within or between CAN regions and subcortical regions only in studies of underweight AN compared to HC (
      • Lord A.
      • Ehrlich S.
      • Borchardt V.
      • Geisler D.
      • Seidel M.
      • Huber S.
      • et al.
      Brain parcellation choice affects disease-related topology differences increasingly from global to local network levels.
      ,
      • Cha J.
      • Ide J.S.
      • Bowman F.D.
      • Simpson H.B.
      • Posner J.
      • Steinglass J.E.
      Abnormal reward circuitry in anorexia nervosa: A longitudinal, multimodal MRI study.
      ,
      • Ehrlich S.
      • Lord A.R.
      • Geisler D.
      • Borchardt V.
      • Boehm I.
      • Seidel M.
      • et al.
      Reduced functional connectivity in the thalamo-insular subnetwork in patients with acute anorexia nervosa.
      ,
      • Haynos A.F.
      • Hall L.M.J.
      • Lavender J.M.
      • Peterson C.B.
      • Crow S.J.
      • Klimes-Dougan B.
      • et al.
      Resting state functional connectivity of networks associated with reward and habit in anorexia nervosa.
      ), but not in studies of weight-restored AN (
      • Cowdrey F.A.
      • Filippini N.
      • Park R.J.
      • Smith S.M.
      • Mccabe C.
      Increased resting state functional connectivity in the default mode network in recovered anorexia nervosa.
      ) as reported here. In contrast, reductions in FC between cortical networks are common in both underweight (
      • Kullmann S.
      • Giel K.E.
      • Teufel M.
      • Thiel A.
      • Zipfel S.
      • Preissl H.
      Aberrant network integrity of the inferior frontal cortex in women with anorexia nervosa.
      ,
      • Geisler D.
      • Borchardt V.
      • Lord A.R.
      • Boehm I.
      • Ritschel F.
      • Zwipp J.
      • et al.
      Abnormal functional global and local brain connectivity in female patients with anorexia nervosa.
      ) and weight-restored (
      • Cowdrey F.A.
      • Filippini N.
      • Park R.J.
      • Smith S.M.
      • Mccabe C.
      Increased resting state functional connectivity in the default mode network in recovered anorexia nervosa.
      ,
      • Lee S.C.
      • Amir A.
      • Haufler D.
      • Pare D.
      Differential Recruitment of Competing Valence-Related Amygdala Networks during Anxiety.
      ,
      • McFadden K.L.
      • Tregellas J.R.
      • Shott M.E.
      • Frank G.K.
      Reduced salience and default mode network activity in women with anorexia nervosa.
      ) AN, though perhaps not as consistently following weight restoration (
      • Boehm I.
      • Geisler D.
      • Tam F.
      • King J.A.
      • Ritschel F.
      • Seidel M.
      • et al.
      Partially restored resting-state functional connectivity in women recovered from anorexia nervosa.
      ,
      • Scaife J.C.
      • Godier L.R.
      • Filippini N.
      • Harmer C.J.
      • Park R.J.
      Reduced Resting-State Functional Connectivity in Current and Recovered Restrictive Anorexia Nervosa.
      ).
      The reduced FC observed in AN following a cardiorespiratory perturbation could be interpreted as a stable consequence of the interoceptive dysfunctions commonly ascribed to this disorder (
      • Pollatos O.
      • Kurz A.L.
      • Albrecht J.
      • Schreder T.
      • Kleemann A.M.
      • Schopf V.
      • et al.
      Reduced perception of bodily signals in anorexia nervosa.
      ,
      • Khalsa S.S.
      • Lapidus R.C.
      Can Interoception Improve the Pragmatic Search for Biomarkers in Psychiatry?.
      ,
      • Jacquemot A.
      • Park R.
      The Role of Interoception in the Pathogenesis and Treatment of Anorexia Nervosa: A Narrative Review.
      )⁠, some of which may persist even in the recovered state (
      • Kaye W.H.
      • Wierenga C.E.
      • Bischoff-Grethe A.
      • Berner L.A.
      • Ely A.V.
      • Bailer U.F.
      • et al.
      Neural Insensitivity to the Effects of Hunger in Women Remitted From Anorexia Nervosa.
      ,
      • Berner L.A.
      • Simmons A.N.
      • Wierenga C.E.
      • Bischoff-Grethe A.
      • Paulus M.P.
      • Bailer U.F.
      • et al.
      Altered interoceptive activation before, during, and after aversive breathing load in women remitted from anorexia nervosa.
      ). However, we believe this to be unlikely for several reasons. First, the described interactions represent state-dependent changes occurring after resolution of an acute perturbation of interoception and were not reflected by substantial group differences in CAN FC at baseline. Second, associations between reduced resting state FC and abnormal interoception and self-representation have been reported previously, though primarily based on cross-sectional data. For example, acutely ill and recovered women with AN have shown decreased activity in the DMN (precuneus), as well as attenuated salience (ACC) and motor network activity relative to HCs (
      • McFadden K.L.
      • Tregellas J.R.
      • Shott M.E.
      • Frank G.K.
      Reduced salience and default mode network activity in women with anorexia nervosa.
      ). Although those decreases were attributed to state-dependent abnormalities of interoception or body image disturbance, such constructs were not manipulated acutely in a state-dependent manner. The current findings therefore represent the first demonstration of a state-dependent alteration of FC showing associations with anxiety, depression, and body image disturbance that is in keeping with, but builds upon, the cross-sectional evidence. Moreover, the lack of BP changes combined with HR increases for AN versus HC in the post-infusion setting may reflect additional evidence of reduced vascular reactivity in AN (
      • Sachs K.V.
      • Harnke B.
      • Mehler P.S.
      • Krantz M.J.
      Cardiovascular complications of anorexia nervosa: A systematic review.
      ,
      • Jenkins Z.M.
      • Phillipou A.
      • Castle D.J.
      • Eikelis N.
      • Lambert E.A.
      Arterial stiffness in underweight and weight-restored anorexia nervosa.
      ), even though we saw no meaningful associations between these physiological indices and the group differences in FC. Finally, based on the observed associations between FC and BSQ scores, abnormalities of body image processing may relate to abnormal interoceptive processing and body image perception in those with AN, and may be particularly influenced by maladaptive frontoparietal-CAN communication. This is an important observation, as interoceptive processing has been hypothesized to be an ingredient involved in the formation of body image perception (
      • Badoud D.
      • Tsakiris M.
      From the body's viscera to the body's image: Is there a link between interoception and body image concerns?.
      ).
      In AN, body image disturbance is prevalent across nearly all stages of the disorder (
      • Favaro A.
      • Santonastaso P.
      • Manara R.
      • Bosello R.
      • Bommarito G.
      • Tenconi E.
      • et al.
      Disruption of visuospatial and somatosensory functional connectivity in anorexia nervosa.
      ,
      • Gaudio S.
      • Riva G.
      Body image in anorexia nervosa: the link between functional connectivity alterations and spatial reference frames.
      )⁠. It contributes to relapse risk (
      • Keel P.K.
      • Dorer D.J.
      • Franko D.L.
      • Jackson S.C.
      • Herzog D.B.
      Postremission Predictors of Relapse in Women With Eating Disorders.
      ), and interoceptive deficits along with a distorted body image have even been proposed as primary drivers of negative eating behaviors (
      • Jacquemot A.
      • Park R.
      The Role of Interoception in the Pathogenesis and Treatment of Anorexia Nervosa: A Narrative Review.
      )⁠. Body image disturbances in weight-restored AN extend beyond self-evaluation to body ownership on experimental tasks such as the rubber hand illusion (
      • Eshkevari E.
      • Rieger E.
      • Longo M.R.
      • Haggard P.
      • Treasure J.
      Persistent body image disturbance following recovery from eating disorders.
      ), a visuoperceptual illusion that is supported by frontoparietal regions (
      • Ehrsson H.H.
      • Spence C.
      • Passingham R.E.
      That's my hand! Activity in premotor cortex reflects feeling of ownership of a limb.
      ,
      • Ehrsson H.H.
      Touching a Rubber Hand: Feeling of Body Ownership Is Associated with Activity in Multisensory Brain Areas.
      ,
      • Gentile G.
      • Guterstam A.
      • Brozzoli C.
      • Ehrsson H.H.
      Disintegration of multisensory signals from the real hand reduces default limb self-attribution: an fMRI study.
      ,

      Guterstam A, Björnsdotter M, Gentile G, Ehrsson H, H. (2015): Posterior Cingulate Cortex Integrates the Senses of Self-Location and Body Ownership. Current Biology. 25:1416-1425.

      ,
      • Karabanov A.N.
      • Ritterband‐Rosenbaum A.
      • Christensen M.S.
      • Siebner H.R.
      • Nielsen J.B.
      Modulation of fronto‐parietal connections during the rubber hand illusion.
      ) but from which amygdala activity/connectivity may protect against (
      • Spengler F.B.
      • Scheele D.
      • Kaiser S.
      • Heinrichs M.
      • Hurlemann R.
      A Protective Mechanism against Illusory Perceptions Is Amygdala-Dependent.
      ,
      • Dobrushina O.R.
      • Arina G.A.
      • Dobrynina L.A.
      • Novikova E.S.
      • Gubanova M.V.
      • Belopasova A.V.
      • et al.
      Sensory integration in interoception: Interplay between top-down and bottom-up processing.
      ). The breadth of the FC changes and their association with body image and affective traits may thus reflect the broader role of interoception in multisensory integration (
      • Kirsch W.
      • Kunde W.
      On the Role of Interoception in Body and Object Perception: A Multisensory-Integration Account.
      ). Our results may also support recent arguments on the neural basis of embodied self-processing networks, which posit that effective coordination of neural activity across self-processing, cognitive control, and salience networks (including the insula and amygdala regions of the CAN) is required for generation of an embodied self-representation (
      • Sui J.
      • Gu X.
      Self as Object: Emerging Trends in Self Research.
      ). This raises the possibility that increased top-down regulation in AN may reflect efforts to self-regulate one’s physiological condition to limit exposure to environmental factors with aversive associations, including food, through disciplined behavior (i.e., food avoidance). We speculate that such a process would likely leverage coordinated communication across numerous networks such as the frontoparietal and CAN networks, but also motor and visual networks, as observed in the present study, and could reflect the identification of large-scale deficits in neuronal synchrony and brain connectivity (
      • Varela F.
      • Lachaux J.P.
      • Rodriguez E.
      • Martinerie J.
      The brainweb: phase synchronization and large-scale integration.
      ) in AN. Pursuant to this view, interoceptively triggered changes in body state in AN individuals would have widespread and disparate effects on emergent properties (
      • Thiebaut de Schotten M.
      • Forkel S.J.
      The emergent properties of the connected brain.
      ) of central autonomic but also cognitive, executive, salience, visual, and even motor networks.
      We observed inverse relationships between CAN FC and self-reported trait measures of body shape representation, depression, and anxiety across groups. These results are consistent with prior reports that activity in CAN regions, such as the amygdala (
      • El Khoury-Malhame M.
      • Reynaud E.
      • Soriano A.
      • Michael K.
      • Salgado-Pineda P.
      • Zendjidjian X.
      • et al.
      Amygdala activity correlates with attentional bias in PTSD.
      ) ⁠or ACC (
      • Paulesu E.
      • Sambugaro E.
      • Torti T.
      • Danelli L.
      • Ferri F.
      • Scialfa G.
      • et al.
      Neural correlates of worry in generalized anxiety disorder and in normal controls: a functional MRI study.
      )⁠, correlates with anxiety traits regardless of group status. Our findings cannot be attributed to effects of acute starvation in AN, given the recruitment of a weight-restored sample, and may thus reflect individually meaningful trait differences across the dimensions of affective and body image concerns. Further insight into the predisposing contributions of these signals is needed, particularly in premorbid samples at risk for development of an eating disorder. Children with avoidant restrictive food intake disorder also show increased risk for other eating disorder diagnoses in adulthood (
      • Bryant-Waugh R.
      Avoidant/Restrictive Food Intake Disorder.
      ,
      • Thomas J.J.
      • Lawson E.A.
      • Micali N.
      • Misra M.
      • Deckersbach T.
      • Eddy K.T.
      Avoidant/Restrictive Food Intake Disorder: a Three-Dimensional Model of Neurobiology with Implications for Etiology and Treatment.
      ), and it remains unclear to what extent interoceptive dysfunction plays a role in the disorder. Our results add to the cross-sectional literature showing relatively decreased cortical FC in both underweight and weight-restored AN (
      • Kullmann S.
      • Giel K.E.
      • Teufel M.
      • Thiel A.
      • Zipfel S.
      • Preissl H.
      Aberrant network integrity of the inferior frontal cortex in women with anorexia nervosa.
      ,
      • Geisler D.
      • Borchardt V.
      • Lord A.R.
      • Boehm I.
      • Ritschel F.
      • Zwipp J.
      • et al.
      Abnormal functional global and local brain connectivity in female patients with anorexia nervosa.
      ,
      • Lee S.C.
      • Amir A.
      • Haufler D.
      • Pare D.
      Differential Recruitment of Competing Valence-Related Amygdala Networks during Anxiety.
      ,
      • McFadden K.L.
      • Tregellas J.R.
      • Shott M.E.
      • Frank G.K.
      Reduced salience and default mode network activity in women with anorexia nervosa.
      ), highlighting the need for investigation into whether it may be a predictive factor for AN in those at risk for developing severe eating disorders. Further, our acute repeated-measures results suggest that an instability of brain-body interactions after adrenergic perturbation may be a part of the noradrenergic pathophysiology of AN (
      • Pirke K.M.
      Central and peripheral noradrenalin regulation in eating disorders.
      ). Potential translational and/or clinical implications of this work could include efforts to develop new laboratory tools or clinical tests capable of delineating interoceptive network dysfunction at the individual patient level. Alternatively, targeting abnormal CAN FC metrics might be incorporated into the development of new treatments focused on the principles of interoceptive exposure, particularly from the standpoint of gastrointestinal processing (
      • Khalsa S.S.
      • Berner L.A.
      • Anderson L.M.
      Gastrointestinal Interoception in Eating Disorders: Charting a New Path.
      ,
      • Zucker N.L.
      • Lavia M.C.
      • Craske M.G.
      • Foukal M.
      • Harris A.A.
      • Datta N.
      • et al.
      Feeling and body investigators (FBI): ARFID division—An acceptance‐based interoceptive exposure treatment for children with ARFID.
      ). Studies investigating the effects of enhancing inter-network communication between the CAN and other self-processing networks may also help to refine the search for new treatment targets capable of addressing anxious and maladaptive body image symptomatology, especially in underweight AN.
      This study has several limitations. The low sample size is balanced by a repeated measures experimental medicine design which allowed us to examine the influence of an interoceptive perturbation on resting brain network activity, and by the paucity of FC studies of AN, most of which have used similar or smaller sample sizes. Another limitation is the subset of CAN regions used for seed-based correlation analysis. We focused on six classically defined cortico-limbic CAN regions linked to sympathovagal control (
      • Benarroch E.E.
      The Central Autonomic Network: Functional Organization, Dysfunction, and Perspective.
      ,
      • Beissner F.
      • Meissner K.
      • Bar K.J.
      • Napadow V.
      The autonomic brain: an activation likelihood estimation meta-analysis for central processing of autonomic function.
      ), however, there is evidence for a ‘complex’ CAN extending beyond the classical regions (
      • Valenza G.
      • Passamonti L.
      • Duggento A.
      • Toschi N.
      • Barbieri R.
      Uncovering complex central autonomic networks at rest: a functional magnetic resonance imaging study on complex cardiovascular oscillations.
      ) that could play a role in the regulation of cardiovascular oscillations during exertion of sympatho-vagal control. Inclusion of other seeds in future studies (e.g., posterior/mid insula) could help to identify additional networks not reported here. We included AN individuals with certain comorbid conditions who were taking selected psychiatric medication but did not think this had a meaningful impact on our findings for several reasons. First, they were stably medicated and screened for recent use of medications (e.g., sedatives) known to acutely modulate fMRI signals. Second, the repeated measures design allowed us to detecting rapid shifts in brain activity that would be unaffected by chronic medication use. Third, most AN individuals have comorbid psychiatric conditions (
      • Hudson J.I.
      • Hiripi E.
      • Pope H.G.
      • Kessler R.C.
      The Prevalence and Correlates of Eating Disorders in the National Comorbidity Survey Replication.
      ), thus findings from our sample have ecological validity for patients receiving treatment in real-world settings and can be supplemented by future studies controlling more tightly for medications and comorbid conditions. Fourth, the strong correlations between trait measures of psychopathology and changes in FC across the whole sample could have been influenced by oversampling both ends of the transdiagnostic spectrum. Balanced sampling will be important for future studies, particularly those seeking to identify group differences in associations between these measures. However, our comparison between groups did not reveal compelling evidence suggestive of group differences in association (see figure S2), and we speculate that the observed relationships reflect neural trait associations along a linear spectrum that serve as a basis for further investigation. Finally, our study sample has two demographic limitations. We recruited an all-female AN sample, which we emphasized to address the far higher (10:1) disorder prevalence rate in females than males. The other is the lack of racial diversity in the AN sample. Since eating disorders are expressed across the spectrum of race and ethnicity (

      Wildes JE, Forbush KT (2015): Ethnicity as a Risk Factor for Eating Disorders. The Wiley Handbook of Eating Disorders, pp 324-337.

      ,
      • Cheng Z.H.
      • Perko V.L.
      • Fuller-Marashi L.
      • Gau J.M.
      • Stice E.
      Ethnic differences in eating disorder prevalence, risk factors, and predictive effects of risk factors among young women.
      ), increasing the diversity of ethnic and racial sampling in future studies will be important in establishing the representativeness of the current findings.
      In conclusion, we found that peripheral adrenergic stimulation induced widespread central autonomic FC reductions in weight-restored individuals with AN compared to HCs. Associations of CAN-FC changes in frontal, parietal, somatosensory, motor, and visual brain regions with trait measures of anxiety, depression, and body image disturbance provide evidence supporting the argument that interoceptive signaling between CAN regions and other brain networks may facilitate the representation and regulation of affective and body image concerns.

      Acknowledgements

      Megan Sinik, BS, Dhvanit Raval, BS, and Chloe Sigman, BS, provided physiological data processing assistance; and Megan Cole, RN, Jeanne Echols, RN, Lisa Augustine, RN, Susan Maxey, RN, and Lindsey Bailey, NT, provided isoproterenol preparation assistance. The authors would also like to acknowledge the key contributions of the late Jerzy Bodurka to the acquisition of the functional neuroimaging data.

      Supplementary Material

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