REVIEWS Functional brain imaging in gastroenterology: to new beginnings Yasser Al Omran and Qasim Aziz Abstract | With more than 100 studies published over the past two decades, functional brain imaging research in gastroenterology has become an established field; one that has enabled improved insight into the supraspinal responses evoked by gastrointestinal stimulation both in health and disease. However, there remains considerable inter-study variation in the published results, largely owing to methodological differences in stimulation and recording techniques, heterogeneous patient selection, lack of control for psychological factors and so on. These issues with reproducibility, although not unique to studies of the gastrointestinal tract, can lead to unjustified inferences. To obtain consistent and more clinically relevant results, there is a need to optimize and standardize brain imaging studies across different centres. In addition, the use of complementary and more novel brain imaging modalities and analyses, which are now being used in other fields of research, might help unravel the factors at play in functional gastrointestinal disorders. This Review highlights the areas in which functional brain imaging has been useful and what it has revealed, the areas that are in need of improvement, and finally suggestions for future directions. Al Omran, Y. & Aziz, Q. Nat. Rev. Gastroenterol. Hepatol. 11, 565–576 (2014); published online 10 June 2014, doi:10.1038/nrgastro.2014.89

Introduction

Centre for Digestive Diseases, Blizard Institute, Wingate Institute of Neurogastroenterology, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, 26 Ashfield Street, London E1 2AJ, UK (Y.A.O., Q.A.). Correspondence to: Q.A. [email protected]

The advent of brain imaging technology in the early 1990s coupled with huge financial investment in brain research has enabled scientists to unravel the mysteries of the human brain.1 Within gastroenterology, it was hoped that brain imaging research on the wellestablished brain–gut axis (reviewed elsewhere2) would serve to identify biomarkers for patients with functional gastrointestinal disorders (FGIDs), and thus help in the diagnosis and treatment of these patients. FGIDs encompass >40 different heterogeneous disorders, including functional dyspepsia, chest pain of oesophageal origin and IBS.3 They are characterized by chronic discomfort, pain and other general symptoms in various locations in the gastrointestinal tract and gallbladder, and occur without any apparent physical, biological or anatomical aetiology. Thus, by definition, FGIDs are system-based disorders, and they share a high comorbidity with other functional pain syndromes such as fibromyalgia and chronic back pain.4 In the USA, FGIDs are estimated to affect 40% of individuals, and cost society more than US$30 billion annually;5 other regions worldwide are similarly affected.6,7 FGIDs are, therefore, among the most prevalent conditions and warrant further research. For FGIDs, patient reports are required to determine symptom-related exacerbations, illness severity and treatment efficacy. Although these patient-reported outcomes can provide useful information, less-biased Competing interests The authors declare no competing interests.

methods such as brain imaging have the potential for greater utility in assessing pathophysiology and treatment efficacy. As brain imaging can provide detail on the gut–brain interaction and the role of cognitive and emotional modulation of afferent inputs, it was initially viewed as a novel and noninvasive method to objectively study brain correlates of peripheral symptoms, response to treatment and a variety of stimuli. Indeed, after its introduction, a myriad of publications relating to brain processing of visceral sensation were published (reviewed elsewhere8,9) and it seemed that visions of identifying disease biomarkers in gastrointestinal d­iseases (particularly FGIDs) would soon be fulfilled. Brain imaging research has greatly enhanced our understanding of the complex interactions between brain areas that process the sensory discriminative, cognitive– evaluative and affective–motivational components of visceral sensation in both health and disease. However, two decades have passed since its introduction and the original expectation of what functional brain imaging in gastroenterology would reveal has not been fully realized, which might in part be related to the nature of the research itself. As FGIDs are heterogeneous, so too are the brain responses to visceral sensation, and thus the translation of findings from functional brain imaging research to the development of biomarkers, formation of a diagnosis or assessment of treatment efficacy might be understandably slow. By contrast, other fields of research have made major advances in the understanding of disease mechanisms and treatment efficacy.10–13 Therefore, why have similar

NATURE REVIEWS | GASTROENTEROLOGY & HEPATOLOGY © 2014 Macmillan Publishers Limited. All rights reserved

VOLUME 11  |  SEPTEMBER 2014  |  565

REVIEWS Key points ■■ Functional brain imaging is an established field in gastroenterology ■■ Although functional brain imaging studies have provided important insights into brain–gut interactions in health and disease, progress in this field has been hampered due to issues with reproducibility and lack of standardization ■■ Reproducibility and standardization issues are not unique to studies of the gastrointestinal tract and are present in other disciplines using functional brain imaging ■■ Adequate control for psychobiological factors, inter-centre collaboration for standardization of imaging and analysis methods, and study of homogenous and psychophysiologically well-characterized healthy individuals could improve reproducibility and standardization ■■ These approaches might provide the crucial first steps towards developing an understanding of the mechanisms of visceral pain in health and disease, and lead to patient-tailored treatments with improved efficacy

Box 1 | Key features of fMRI and PET fMRI ■■ Measures brain activity by detecting changes in regional cerebral blood flow, volume and oxygen saturations. In particular, BOLD fMRI is dependent on the paramagnetic properties of blood haemoglobin that enables it to identify regional distribution related to changes in neuronal activity.16,98 ■■ Uniquely noninvasive. fMRI provides reasonably high spatial and temporal resolution compared with other imaging modalities and is suitable for longitudinal studies in large numbers of patients. These features have been particular advantageous in pharmacological studies.99,100 ■■ Some physiological restraints. For example, the haemodynamic response takes place over several seconds and varies to some extent across tissues. With fMRI, a resolution of a few seconds can be expected.101 Furthermore, in situations in which the neurovascular coupling might be compromised (for example, by drugs or anaesthesia), the detected temporal response can be very different to the true neuronal activity.102,103 Likewise, the spatial resolution of BOLD fMRI is limited by the resolution of MRI images; although typical spatial resolution are millimetres apart, MRI images work at a resolution ten times greater, giving low signal:noise ratio.98 PET ■■ PET is based upon the principle of using positron-emitting radioisotopes to measure particular biochemical and physiological processes.104,105 ■■ Incorporation of radioisotopes into pharmaceuticals following intravenous administration, to enable the imaging of their uptake, makes PET one of the most sensitive means of molecular imaging, helping to determine receptor-binding sites and signalling systems.106 ■■ Due to the risk of radiation exposure, PET studies are not used routinely. Longitudinal studies are usually not performed unless there is a substantial gap between consecutive studies. ■■ Radioactive ligands are expensive and there are only a handful that work in vivo, thus there can be a difficulty in generating a relevant ligand within the context of the experiment.107 Abbreviations: BOLD, blood-oxygenation-level-dependent contrast imaging; fMRI, functional MRI.

advances not been seen in brain imaging research, particularly related to gastroenterology? The time for reflection has come at an opportune moment, with the development of The Human Brain Project and the Brain Research through Advancing Innovative Neurotechnologies i­nitiative, which are aimed at further­ing our understanding of the human brain.14,15 These initiatives will hopefully enable brain imaging research in gastroenterology to match that of other areas of medicine. This article reviews the ways in which functional brain imaging in the field of gastroenterology has developed since its ­initiation, paying special attention to FGIDs; it also brings to the reader’s attention particular areas that are still in need 566  |  SEPTEMBER 2014  |  VOLUME 11

of improvement and suggests how future improvements might be achieved.

Traditional brain imaging techniques PET and fMRI Although many techniques are available to measure and image human brain activity, the two most extensively used are PET and functional MRI (fMRI). They in­directly map and localize brain activity by imaging regional metabolic or physiological effects of modulated effects of brain activity. Although it is beyond the scope of this Review to provide extended discussion of brain imaging techniques, a brief overview is useful to appreci­ ate both the issues and the merits of these techniques within fu­nctional brain imaging research (Box 1). What have these techniques revealed? Despite the technical drawbacks of fMRI and PET, they have helped unmask different aspects of gut–brain interactions. As there are a plethora of studies published on the topic, the following section reviews fMRI and PET studies that form the basis of the inconsistencies seen in the field, as well as areas that have been elaborated upon by the latest studies. For more extensive reviews, readers are referred elsewhere.9,16 Homeostatic–afferent network Pain has been described as a ‘homeostatic emotion’ that is different from other senses in that it is multi­dimensional and complex.17 When patients are subjected to nocicep­ tive stimuli, an array of cortical, subcortical and cerebel­lar structures are consistently activated, and these structures have been collectively referred to as the pain homeostatic afferent network or ‘pain neuromatrix’18,19 (Figure 1a). With regards to a more-specific visceral homeostatic– afferent network, the regions most consistently activated by either rectal, oesophageal, colonic or gastric stimulation in healthy individuals are the anterior cingu­ late cortex (ACC) and the insula (together, they have a major role in the conscious experience of pain), followed by the primary somatosensory cortex, prefrontal cortex, the posterior parietal cortex and thalamus.9 This pattern of activation is also seen in patients with FGIDs, except with greater activity in certain areas such as the insula and ACC.20–22 The fact that somatic pain studies show similar levels of activity in these specific brain regions supports the validity of this intricate neuronal network for pain processing. To prevent confusion, it must be noted that the latest studies have reported that the regions involved in the pain neuromatrix are not exclusive to pain processing, but serve many other functions.23–25 The term is, therefore, now considered a misnomer, and might have formed the basis of some inconcistencies in the field. Its use cannot therefore be fully justified. Brain responses to visceral and somatic pain Despite the similarity between visceral and somatic pain studies in the activation of homeostatic brain regions, experimentally evoked visceral pain is characterized as



www.nature.com/nrgastro © 2014 Macmillan Publishers Limited. All rights reserved

REVIEWS a

Homeostatic afferent network

b

Emotional arousal network

aMCC

aMCC spgACC

pgACC Insula

Brain stem

Thalamus

Amygdala

Figure 1 | Networks of brain activation in visceral stimulation studies. a | The homeostatic–afferent network encompasses areas that are thought to resemble those of pain when stimulated in visceral pain studies both in participants with a FGID and in participants who are otherwise healthy. The core regions are shown. b | The emotional arousal network is depicted. Cognitive (e.g. expectation and anticipation), emotional (e.g. sadness) and psychological aspects have all been shown to be involved in visceral perceptions and this progress has established what is known as the ‘emotional arousal network’. The central components of this network are the amygdala and parts of the ACC. Abbreviations: ACC, anterior cingulate cortex; aMCC, anterior midcingulate cortex; FGID, functional gastrointestinal disorder; pgACC, perigenual ACC; sgACC, subgenual ACC.

being more diffuse, variable and unpleasant than cutan­ eous pain,26 probably because of neuro­anatomical differences between the two types of pain, which brain imaging studies have since helped to further characterize. In a series of experiments whereby healthy participants were exposed to both visceral mechanical and cutaneous thermal nociceptive stimuli of similar intensity, it was shown that cutaneous thermal stimuli induced increased activity in the ventrolateral prefrontal cortex and the bilateral anterior insula, whereas visceral mechanical stimulation (in the same dermatome) corresponded with increased bilateral activity in the primary motor cortex, primary somatosensory cortex and the dorsal ACC.26–28 Brain processing of sensation evoked by stimulation of the smooth muscle (rectum and distal oesophagus) and striated muscle (anus and proximal oesophagus) parts of the gastrointestinal tract have also shown similar differences.29 Differences in processing of visceral and somatic sensations have also been demonstrated at the level of the brainstem with moderately painful, electrical stimulation of the rectum (visceral) or the lower surface of the abdomen (somatic) being associated with increased responses in the nucleus cuneiformis and the periaqueductal grey, but with greater activation in the nucleus cuneiformis in visceral than somatic stim­ u­lation.30 These differences in brain activation patterns between visceral and somatic pain are probably due to differences in the peripheral neuroanatomy of painspecific fibres and pathways, which lead to differences in the quality of s­ensation perceived and, in turn, brain activation patterns.30 Psychobiological aspects of visceral sensation As psychobiological aspects (stress, personality and mood) are all known to modulate the pain experience,31,32 pain might be as much psychological as it is (patho) physiological. This concept was originally depicted in somatic pain studies, in which emotional factors, such

as unpleasantness, emotional traits (aggressiveness, lovingness, protectiveness, and so on) and positive or negative mental states were shown to have a huge effect on the motivational aspects of the pain experience.33–35 In FGIDs, psychological disorders such as dysthymia, depression and heightened anxiety have been reported,5 which prompted the investigation of the effects of different emotional states on the intensity and distribution of cortical activation. Through such investigations, an affective–motivational pain circuitry has been established, which includes the amygdala and ACC sub­regions (both of which form the central components of this circuit) and is colloquially referred to as the emotional arousal network36 (Figure 1b). The involvement of the anterior insula hippocampus and the dorsal pons forms the extended emotional arousal network.37,38 The study of a negative emotional state is a good example of the use of brain imaging studies to understand the workings of this network. For instance, oesophageal distension during a negative emotional state was associated with increased cortical activation of the dorsal ACC and the anterior insula, and was perceived more intensely and generated greater anxiety than distension in a neutral emotion state (elicited by presenting neutral facial expressions).39 Additionally, exposure to oesoph­ ageal distension and music to induce negative emotional states also resulted in increased activity in the right ACC and anterior insula on fMRI.40 These findings highlight that emotional factors have a profound effect on patterns of cortical activity. As many patients with a FGID report feeling depressed, anxious and associate gastrointestinal sensations with negative feelings,41 these findings establish the importance of controlling for emotional factors in brain imaging studies in both health and FGIDs. Cognitive factors, in particular anticipation, perception and attention–distraction, are other important psycho­biological components and so might play a part in awareness of visceral sensation. Studies have shown that the intensity of pain can be attenuated when an individual is distracted, and heightened when focusing upon the painful stimulus.42 In FGIDs, brain imaging studies have demonstrated that patients might be more attentive to their visceral sensations, which could, in part, be responsible for their persistent symptoms.43 Additionally, most gastrointestinal sensations are not consciously perceived in health, in contrast with other modalities such as touch, which might be partly due to decreased selective attention compared with other modalities. In fact, a series of experimental studies provided evidence that selective attention to visceral sensation increases brain activity in relevant areas, which could exaggerate perception of visceral sensations and lead to increased emotional responsiveness. 44,45 Potentially, these processes can occur in FGIDs, and could explain the amplified sensory awareness observed in these patients.43 The aforementioned studies provide convincing evidence for the effect of psychological factors on visceral pain, however, these studies were conducted mainly in healthy individuals. To provide further insight, brain

NATURE REVIEWS | GASTROENTEROLOGY & HEPATOLOGY © 2014 Macmillan Publishers Limited. All rights reserved

VOLUME 11  |  SEPTEMBER 2014  |  567

REVIEWS Box 2 | Methodological variations in functional brain imaging studies Study population There can be huge heterogeneity between the individuals included in studies; they might differ in age, sex, ethnicity and pain thresholds. Participants can also have psychiatric or pain-related comorbidities, which if not screened for might cause variations in the results between functional brain imaging studies.56,108 Technical issues Each laboratory can have different ways of conducting their studies. For instance, the choice of brain imaging technique, the choice of scanner, the protocol design and a plethora of statistical analysis decisions.70 Cumulatively, these decisions can heavily influence results. Method of stimulation There are many forms of stimulation (e.g. mechanical, electrical, thermal), and depending on which one is used, each can activate different supraspinal regions. Studies can also use different stimulus intensities (subliminal, nonpainful or painful), which can be intermittent or be incrementally more painful. Studies can also subject participants to repeated stimulus exposure.54 Number of study participants Although a number of studies have shown statistically significant brain activations, some have used relatively small sample sizes and might have been underpowered.9 Greater numbers are required to determine verified patterns of brain activation following visceral stimulation.

imaging studies of visceral pain in patients with a FGID have also been conducted. The first comparison of g­­astrointestinal-associated cortical activity between healthy individuals and patients with FGIDs used PET and implied that activation of the ACC was attenuated in IBS, but activation of the prefrontal cortex was heightened.46 The reduced activity in the ACC was originally thought to be because of aberrant modulatory effects of the endogenous opioid system, whereas enhanced activity in the prefrontal cortex was thought to be because of increased awareness of gastrointestinal stimulation. Findings from Wilder-Smith et al.47 supported the notion of endogenous pain modulation. By using hetero­topic stimulation (that is, two distant but simultaneously occurring painful stimulations), they demonstrated that noxious stimulation of the foot reduces rectal pain during balloon distension in healthy individuals as controls, but increased pain in patients with IBS. Specifically, healthy individuals demonstrated a bilateral activation of supraspinal structures, including the ACC and the ventral and dorsal prefrontal cortex;47 activity in these areas was reduced in patients with constipation-predominan­t IBS and absent in those with diarrhoea-predominant IBS. More­­over, controls showed decreased activity in areas of the homeostatic–afferent network, whereas these regions showed no change or showed increased activity in patients with constipation-predominant IBS and diarrhoea-predominant IBS, respectively, as well as showing increased activity in the amygdala and orbitofrontal cortex, which are pain modulatory regions.47,48 Other studies in IBS using heterotopic stimulation have validated the altered brain activation in these regions.49 Brain activity as a biomarker? As detailed previously, the homeostatic–afferent network seems to be activated during visceral stimulation both in healthy controls and in patients with FGIDs. However, 568  |  SEPTEMBER 2014  |  VOLUME 11

studies do provide contradictory evidence and suggest the saliency of this network in the processing of all homeostatic information and not just pain. The ACC is a brain region that is activated by a range of emotional states and seems to facilitate prefrontal influences associated with corticolimbic inhibition, emotion modulation and cognitive factors of pain. Although the ACC is one of the most consistently activated brain regions of the homeostatic–afferent network following visceral stimulation,9 some studies in patients with IBS show greater activity whereas others show reduced activity compared with healthy controls.21,47,50–54 These differences were interpreted as being related to possible antinociceptive effects of pathways arising from the ACC, disturbances of the emotional and associative processing of visceral sensation or to altered sensitization of pain pathways. Thus, not only are there disparities between the results of brain imaging studies, but there are also different hypotheses to explain these disparities. This confusion is exemplified further by fMRI studies that demonstrate greater activity in several brain regions (including those relating to sensory as well as to cognitive and emotional regions) in patients with IBS than in age-matched and sexmatched controls when subjected to colorectal pain.51 One interpretation of these data is that the increased visceral pain in IBS might be due to increased afferent processing, perhaps because of increased sensitization of afferent pathways by previous infectious or inflammatory events. However, increased selective attention to the visceral stimulus can also explain this increased activity in supraspinal structures, as has been suggested in other studies.44,45 Somatosensory, cognitive and affective factors that modulate pain are probably all intertwined, and thus alteration in one factor alters another. For instance, if more attention is paid to pain, then there might automatically be greater processing of the corresponding sensory area. As a result, it might not be possible to disentangle the various factors involved in pain processing in patients with FGIDs by fMRI or PET alone, because these techniques have neither the temporal nor spatial resolution to do so.

Methodological variations

Conceivably, variations in methodology and patient selection might explain the results of brain imaging studies rather than the findings being mechanistically important. These variations have been previously identified9 and are summarized in Box 2. These variations can have substantial effects on study conclusions. Using sample size as an example and with regards to the variability with ACC activity as described in the previous section, it might be possible that some study participants could be claustrophobic and anxious whilst lying inside a noisy MRI scanner, and perhaps made more anxious by a balloon about to be painfully inserted into their rectum and inflated. As a result, the ACC of those patients might be more active throughout the length of the study. Fear and anxiety can exaggerate and thus, if too few individuals are included in a study, can act to produce erroneous results in subtraction analysis. By using larger sample



www.nature.com/nrgastro © 2014 Macmillan Publishers Limited. All rights reserved

REVIEWS sizes, such unspecific brain activation can be limited, and more accurate results might be achieved.

Investigating and questioning study design Meta-analyses As the aforementioned methodological differences between brain imaging studies are problematic, powerful quantitative meta-analytical techniques are being undertaken to collate results and to form conclusions as to the most accurate representation of physiological supra­spinal responses after visceral stimulation.55,56 For example, a meta-analysis that examined studies involving supra­ liminal rectal balloon distensions suggested that there is greater engagement of regions linked to emotional arousal and homeostatic afferent processing in patients with IBS than in healthy controls, which supports the notion of a central nervous system deregulation in IBS.56 Thus, these meta-analytical techniques are incredibly useful in elucidating which brain imaging studies have used the better methodological parameters (for example, study design or analytical methods) and have further supported the idea that these parameters are the main reason for the variable results in brain imaging studies as opposed to an intrinsic limitation of investigational approaches.56 Critical appraisals Critical appraisals can be another approach to unravel possible reasons for inter-study differences. For example, using subliminal rectal stimulation of increasing intensity (that is, pressures of 10 mmHg, 15 mmHg and 20 mmHg) coupled with fMRI, Lawal et al.57 aimed to investigate supraspinal activity independent of stimulus-related cognitive modulation in patients with IBS compared with age-matched controls. They reported substantially greater supraspinal activity in patients with IBS than controls irrespective of stimulus-related cognitive processes, perhaps indicating the presence of increased afferent signalling within supraspinal circuits.57 However, in a critical appraisal, it has been argued that there might be alternative explanations for such a response.58 For one, patients with IBS can show a global hypersensitivity to a wide range of stimuli59 and, as discussed previously, hypervigilance (as a result of increased attention) can also play a part. Thus, both forms of hyper-responsivene­ss seem more related to central mechanisms of signal amplification as opposed to signal intensity. Moreover, a wealth of studies have shown that the anticipation of visceral stimulation, regardless of whether stimulation actually followed, can cause activation of specific brain regions, which include the primary and secondary somato­ sensory cortices, midcingulate cortex, and the posterior parietal cortex.60–62 Taken together, although the chosen study design undertaken by Lawal et al. 57 aimed to minimize cognitive alteration of visceral afferent signal transmission, it might have actually engaged processing related to hyper-responsiveness and uncertain expectation. Furthermore, the complete removal of emotional and cognitive stimulus processing in humans to study pure visceral afferent signal transmission could only be ­possible if individuals were anaesthetized.58

Another example in which critical appraisal can be helpful comes from a study of the role of gut microbiota in altering human brain activity.63 The study was conducted in light of evidence that suggests the enteric microbiota might have an overwhelming influence on normal human behaviour, and that alterations in its composition, or its metabolic products, can, in part, be responsible for the pathophysiology of psychiatric illnesses or FGIDs.64,65 In this novel study, healthy women received either a fermented milk product probiotic (FMPP) beverage made up of five bacteria, or a non­ fermented milk product (control);63 fMRI was conducted before and after a 4‑week course of the products and all participants were exposed to an emotional attention task. Compared with the control, consumption of the FMPP beverage resulted in alterations in processing by cortical and subcortical regions, and caused reduced reactivity to the emotional attention task in brain regions involved in the processing of somatic and visceral sensory information and that are altered in conditions such as anxiety and IBS. 63 However, there are two key limitations of this study. Firstly, stool samples were analysed preinterventio­n and post-intervention, and there was no statistically significant change in microbiota composition between the two groups. It might therefore be possible that the bacteria in the FMPP beverage did not survive transit through the gastrointestinal tract. Secondly, the study authors did not provide any information regarding the behavioural responses to the emotional task and, therefore, it is not possible to determine whether the participants attended to the task differently after the intervention. Taken together, the above studies highlight the importance of using psychophysiological and b­ehavioural data to support any conclusions made.

Multicentre collaborations A possible solution to improving the quality of functional brain imaging studies might be to develop multicentre partnerships with standardized protocols to enable comparable results and the pooling of these results between centres. This tactic would enable clearer conclusions and potential confounders to be controlled for more appropriately. Such an approach is now being undertaken, with a number of initiatives, namely the Genes in Irritable Bowel Syndrome Research Network Europe (GENIEUR) and the Pain and Interoception Imaging Network (PAIN), being developed to support such an international partnership.66,67

Interpretation of studies Inferences In addition to the limitations described above, another major limitation of brain imaging studies is as a result of inferences. These come in two forms, forward inferences and reverse inferences (Box 3), but they can lead to inappropriate conclusions being drawn. 68–70 The homeostatic–afferent network is a prime example of such inappropriate ‘use’ of inference. Despite its name, the colloquial term of pain neuromatrix is a misnomer, as its structural components are activated by a range

NATURE REVIEWS | GASTROENTEROLOGY & HEPATOLOGY © 2014 Macmillan Publishers Limited. All rights reserved

VOLUME 11  |  SEPTEMBER 2014  |  569

REVIEWS Box 3 | Types of inferences in functional brain imaging studies Both forward and reverse inferences have been used in functional brain imaging studies. Forward inferences are data-driven approaches that evaluate patterns of brain activation during different conditions to form conclusions as to mechanisms for the pattern seen. For example, in a study whereby individuals are exposed to a painful stimulus and a nonpainful stimulus, the brain regions that are more active after a painful stimulus might be inferred to have a greater role in the processing of pain than the regions that are less active. The problem with such an approach is that it is essentially correlational in nature, as (in the example) the activated brain regions, might be activated as a result of anticipation or fear of pain as opposed to pain itself. Whether supraspinal structures that are activated during the different mental and/or perceptual processes are required for the occurrence of those processes might therefore be difficult to determine.68 A reverse inference is the reverse processes, that is, affirming the involvement of a particular brain region in the present study based on acquired knowledge from past research. It can also be a relatively weak approach as there is mass activation (by a wide range of cognitive tasks) across the brain regions; thus, activation of a particular region is predominantly unselective.69,70

of different sensory stimuli and cognitive settings, and not solely by nociceptive stimuli.24 When Melzack conceptualized the original ‘neuromatrix’ in 1989, he referred to a network that produces an output pattern or ‘­neurosignatures’, and that pain comprises one of many neuro­signatures created by the neuromatrix. The activation of the homeostatic–afferent network, therefore, cannot explicitly translate to the recognition of pain in the brain. In support of this view, a landmark study using fMRI has demonstrated that nociceptive, visual, auditory and tactile stimuli can generate spatially indistinguish­ able responses in the ACC, the insula and the largest part of secondary somatosensory cortex, denoting that the majority of the brain responses to nociceptive stimuli are a result of multimodal neural activation (that is, activation that can be elicited by any kind of stimulus independently of sensory modality).71 These findings were validated further by a study using electroencephalo­ graphy that generated similar results. 72 Within this context, perhaps reverse inference can act as a fallacy. If a conclusion is drawn based on the previous understanding of the role of the homeostatic–afferent network in pain processing—for example, it has been previously shown that if a person feels pain, then the homeostatic– afferent network is activated and therefore this person is experiencing pain—then this conclusion is flawed, as other cognitive processes can activate this network.73 The only way in which such inferences might be appropriate is when a certain amount of logistic regression analysis is applied. In other words, we must know what else activates the homeostatic–afferent network; if these elements are controlled for, then reverse inferences could be a powerful technique.74

Multivariate analyses Multivariate techniques, such as multivariate pattern analy­sis (MVPA), are good examples of allowing us to make more substantiated inferences. MVPA denotes a particular set of methods that analyse neural responses, elicited by certain stimuli or during certain tasks, as patterns of activity. As a result, it enables investigation of the multiple brain states that a cortical region or system 570  |  SEPTEMBER 2014  |  VOLUME 11

might be in, and thus is much more sensitive than the conventional analysis techniques in unveiling withinregion spatial differences in brain activity throughout different conditions.75 Using this form of analysis, Liang et al.76 showed that for any region of the human primary sensory cortices—namely the primary somatosensory, primary auditory and primary visual cortices—the spatial pattern of fMRI responses elicited by sensory stimuli from different modalities are adequately distinguishable to enable discrimination of that modality (for example, discrimination between auditory and tactile stimuli using the fMRI responses sampled within the primary visual cortex). They further demonstrated that two stimuli of the same modality also evoke distinctive fMRI responses in the specific primary sensory cortex (for example, two tactile stimuli to the two separate fingers evoke distinguishable fMRI responses in the visual cortex). These results suggest that both transient and isolated stimuli of a particular sensory modality do not activate the same ­neuronal populations as those of other sensory modalities. In the context of pain research, these results indicate that a specific signature for pain might exist and can be distinguished from the neural activity elicited by other stimuli.76 Furthermore, as MVPA is performed using the same, normalized blood-oxygen-level-dependent contrast imaging (BOLD) signal responses, the results described here could not be due to differences in mean amplitude of the signal within each primary sensory cortex, but must be due to spatially distinct stimulus-specific BOLD signals. The use of reverse inference here, therefore, might be appropriate, and in this case could demonstrate how sensory information, including that from the gastrointestinal tract, is coded in the brain.76 Taken together, researchers should use inferences to guide succeeding brain imaging and behavioural studies only if they undertake appropriate measures to control for confounding factors to improve the validity of their conclusions.

Getting back on track

In the previous sections, we have summarized what we think are the main problems that have hindered the progress of brain imaging studies within gastrointestinal research. Although some areas have already undergone natural improvement (for example, using more appropriate sample sizes), other problems still need to be addres­ sed. Fortunately, some of these problems could be cor­­­rected with minor modifications and by u­ndertaking other approaches.

Connectivity Although the concept of the homeostatic–afferent network might have been distorted from its intended meaning, one theory that has been proposed is that instead of being a simple enumeration of areas of activation, the pattern of activation of the various structures in the network, as an ensemble, is responsible for the processing of pain.71 This concept might be a better explanation as it provides an improved understanding of the differences in the activation of the ACC as discussed earlier (that is, it is the pattern of activation, not



www.nature.com/nrgastro © 2014 Macmillan Publishers Limited. All rights reserved

REVIEWS Pain cluster 1 Baseline ■ High neuroticism ■ High anxiety ■ High sympathetic tone ■ High cortisol release ■ Low parasympathetic tone

In pain ■ Tolerate less pain ■ Habituate less ■ Sympathetic withdrawal ■ Parasympathetic activation

Pain cluster 2 Baseline ■ High extroversion ■ Low anxiety ■ Low sympathetic tone ■ Low cortisol release ■ High parasympathetic tone

In pain ■ Tolerate more pain ■ Habituate more ■ Sympathetic activation ■ Parasympathetic withdrawal ■ Preferential activation of right medial/frontal cortex and right anterior insula

Figure 2 | Pain clusters after visceral stimulation. Integration of cognitive and psychological processes, which contribute to the intrinsic heterogeneity of chronic visceral responses, is in need of development in the field of gastroenterology. Integration involves multimodal approaches that aim to measure different psychobiological aspects as well as functional brain imaging to provide insights into physiological and pathophysiological states. Using such approaches, two pain clusters were identified in the general population and each had different responses.

the individual activation of the ACC), and parallels the findings of Liang and colleagues.76 As a result, there has been a shift from analysis of brain regions to analysis of networks in what is known as connectivity analysis; this approach has also been implemented to characterize the emotional–arousal network in both patients with IBS and healthy individuals. For instance, connectivity results revealed that sex differences in supraspinal responses in patients with IBS might be due to modifications of the emotional–arousal network rather than visceral afferent processing.38,77 Moreover, alterations in serotonin signalling within the gut–brain axis have been implicated in the pathophysiology of IBS. Using connectivity analysis, patients with IBS, compared with healthy controls, showed a greater engagement of the emotional–arousal network, with dysregulated serotonergic signalling of this network having a part to play in central pain amplification and IBS pathophysiology.78 This paradigm shift towards connectivity analysis has the potential to help clarify certain behavioural and clinical features of FGIDs, such as hypervigilence, sex differences and anticipation. But, more importantly, connectivity analysis can act as a surrogate end point, a biomarker that is intended to substitute for a clinical end point and is expected to predict clinical benefit.79,80 Indeed, certain patterns of activity might be seen in disease processes, which could foster the development of other treatments for FGIDs and potentially other chronic pain disorders. However, this progress will only happen if confounders are controlled for adequately. If this level of control is not achieved, then no matter how advanced the connectivity analysis tool is it will fail.

Resting-state fMRI Brain activity is present even in the absence of an extern­ ally activated task, which might cause fluctuations in the

BOLD signal. Thus, when experimental parameters are applied, this background brain activity can distort the BOLD signal, making it difficult to elucidate the brain mechanisms involved within the study. Resting-state fMRI is a fairly new approach that could resolve this issue. Rather than evaluate task-related brain activity or areas activated via stimulation, resting-state fMRI is conducted to examine brain activity independent of stimulation or performing a certain task.81 This approach can be used to explore the brain’s functional neural network in health and how it might be altered in pathology. As each individual has subtle differences in neural organisation, reproducibility and comparison of results can be a concern. However, in a study published in 2010, r­e sting-state fMRI data from 35 independent inter­ national centres, which included >1,400 individuals, was gathered and collectively showed consistent networks of brain activity, suggesting that independent restingstate fMRI datasets can be collected, shared and compared,82 and resting-state fMRI is a reliable technique. But what is the value of exploring functional neural networks using this approach? Although the search for biomarkers and surrogate end points has salience in the biomedical field, the search for ‘endophenotypes’ is ongoing in the field of psychiatry. Both biomarkers and endophenotypes have similar functions in their respective fields—endophenotype­s being defined as “measurable components unseen by the unaided eye along the pathway between disease and distant genotype”.83 Thus, information about functional neural networks (by means of resting-state fMRI) could provide endophenotypes for disease vulnerability. This theory is supported by reports of altered functional neural networks in neuropsychiatric disorders84 and in pain conditions,85 and thus has notable promise in FGIDs that are associated with pain and mood changes. For instance, using resting-state fMRI, the authors demonstrated that female patients with IBS had greater dysregulation in insula-centric networks that are involved in emotional arousal detection and sensori­ motor processing than their male counterparts.86 This study was the first large-scale resting-state fMRI analysis performed in patients with IBS to further characterize sex differences in the condition, and highlights the potential that r­esting-state FMRI can yield in the field of gastroenterology.

The need for integration: psychobiology Another major area in need of improvement in the field of gastrointestinal science is the integration of psychobiological processes. Despite being known to be involved in processing of visceral stimuli,87 psychobiological processes have not been incorporated into the method­ology, design and results from most brain imaging studies, nor with other branches of neuro­science such as cognitive and psychiatric neuroscience that have u­tilized brain imaging. By contrast, somatic pain research has made substantial headway in this regard by trying to take into consideration and integrate cognitive (for example, expectation and anticipation), emotional (for example, sad­ness) and psychological aspects into

NATURE REVIEWS | GASTROENTEROLOGY & HEPATOLOGY © 2014 Macmillan Publishers Limited. All rights reserved

VOLUME 11  |  SEPTEMBER 2014  |  571

REVIEWS Table 1 | Alternative techniques to aid understanding of the brain–gut axis in health and disease Technique

How it works

Benefits

Limitations

Study findings

Magnetoencephalography

Detects the magnetic fields arising from postsynaptic neurons

Has a much better temporal resolution than fMRI or PET

Insensitive to radial currents (that is, activity arising from deeper brain sources), and so these currents are difficult to measure

The existence of a temporal synchrony in the human cortex during human swallowing behaviour109 A functional diversity in the somatosensory cortices (that is dependent on temporal and frequency dynamic) in response to oesophageal stimulation110 The combined use of magnetoencephalography with other brain imaging techniques is possible111

Electroencephalography

Measures ionic current flows, through noninvasive scalp electrodes

As with magnetoencephalography, electroencephalography has excellent temporal resolution

As with magnetoencephalography, electroencephalography is insensitive to radial currents

A possible short-term sensitization of the oesophagus can elicit central cortical reorganization112 Painful visceral stimulation in patients with chronic pancreatitis might modify cortical projection of the nociceptive system, and could lead to impaired habituation responses113,114

Arterial spin labelling

A noninvasive fMRI-based technique that allows for the measurement of cerebral blood flow through the use of magnetically labelled arterial blood water, which acts as an endogenous, freely diffusible tracer

Has exquisite lowfrequency sensitivity, and so can be used to detect quantitative, baseline, changes in cerebral blood flow in conditions with a tonic stimulus, such as in chronic pain states like FGIDs115

The temporal resolution and sensitivity is generally lower than BOLD fMRI in detecting stimulus-dependent or task-dependent activity

Its use might be appropriate in imaging studies requiring longer time scales and involving psychobiological elements such as behavioural states and traits116,117 The possible diverse roles of the insula and midcingulate cortex in pain processing in a model of muscular pain118

DTI

An MRI technique that uses the diffusivity of water as a probe to infer the anatomy and integrity of different white matter tracts

The anatomical information obtained about neural structures enables better validated inferences

Issues relating to the direction of motion of water molecules, and the sensitivity of motion and scanning time119

The diffusion response in DTI might be more closely linked to neuronal activity than BOLD fMRI (and so could help overcome some of its limitations)120 Patients with FGIDs have microstructural changes in neural structures, suggesting that FGIDs, despite the name, might have an organic and structural component 95–97

Structural MRI

Morphometric techniques measure the volume or shape of grey matter structures

As with DTI, the anatomical information obtained about neural structures enables better validated inferences

Provides only structural information and not any functional information

Patients with IBS,121,122 painful chronic pancreatitis,123,124 and even chronic back pain125 have shown disease-related modification in the grey matter and/or cortical regions

Abbreviations: BOLD, blood-oxygen-level-dependent contrast imaging; DTI, diffusion tensor imaging; FGID, functional gastrointestinal disorder; fMRI, functional MRI.

experimental design, and to investigate how they might affect pain processing. Therefore, similar integration of other brain imaging neuroscience modalities into visceral pain studies could be equally fruitful and should be adopted, and might help demystify the puzzling and heterogeneous nature of FGIDs.87 In striving to achieve this goal, it will be necessary to delve into other uncharted areas within the field of gastroenterology, in which psychobiological processes, functional brain imaging and neurophysiology can all be integrated. Efforts to undertake such levels of inte­ gration are underway. For example, although early studies have highlighted how cognitive and affective factors can modulate visceral pain, 39,40 there is still a paucity of comprehension of how inter-individual differences in psycho­biological aspects influence visceral pain perception, especially in a more chronic setting such as in certain personality traits. In a study investigating how neuroticism might influence brain processing of visceral pain, Coen et al.88 reported a positive correlation between neuroticism and brain activity in areas associated with the cognitive and emotional processing of pain during the anticipation of pain; when pain was experienced, neuroticism negatively correlated 572  |  SEPTEMBER 2014  |  VOLUME 11

with these regions. This finding could reflect a mal­ adaptive response in neurotic individuals, such that there is an overarousal during the anticipation of pain, and avoidance during pain.88 Furthermore, the same group expanded on this area by examining the existence of d­istinct human pain clusters, which are comprised of discrete psychobiological and genetic profiles linked with variations in the processing and perception of pain. 89 They reported two reproducible pain clusters. The first (pain cluster 1) can be described as being more neurotic, with greater baseline sympathetic tone and higher cortisol release than the other pain cluster, and an over-representation of the S allele of the serotonin transporter-linked polymorphic region (5-HTTLPR), and associated with alterations in pain sensitivity, pain modulation and responses to analgesia.90–92 Individuals in pain cluster 1 had increased parasympathetic tone to pain, and had lower pain thresholds and habituated less to pain. The second pain cluster (pain cluster 2) has the opposite profile at both baseline and during pain89 (Figure 2). Similar pain clusters have been further identified specifically in the context of functional chest pain, with cluster 2 being overrepresented in patients.93 Although the clinical utility



www.nature.com/nrgastro © 2014 Macmillan Publishers Limited. All rights reserved

REVIEWS Box 4 | Approaches to improve functional brain imaging studies in gastroenterology Study design ■■ Avoid unsubstantiated inferences as much as possible in forming hypotheses ■■ Use of appropriate study population and size ■■ Screen for possible cognitive and emotional confounders ■■ Use of multimodal imaging modalities, including both functional (e.g. EEG, ASL, fMRI) and anatomical (e.g. DTI, structural fMRI) Time independent ■■ Use of meta-analyses, critical appraisals and literature reviews ■■ Inter-centre collaborations Study analysis ■■ Use of advanced analytical techniques (e.g. connectivity analysis, MPVA) ■■ Make inferences if validated following extensive analysis Some of these interventions require minimal effort and are needed to improve the quality of studies in gastroenterology. Please note that only suggestions made in this article are shown and other, potential suggestions might also be considered. Abbreviations: ASL, arterial spin labelling; DTI, diffusion tensor imaging; EEG, electroencephalography; fMRI, functional MRI; MVPA, multivariate pattern analysis.

of identifying pain clusters remains to be seen, it has the potential to improve homogeneity not only in brain imaging research, but also in drug trials, which have yet to succeed in identifying similar cohorts of participants for study.94 Taken together, the preceding studies are prime examples of ways in which integration of different disciplines into brain imaging research might be undertaken, and could help to guide future brain imaging studies in the field of visceral pain research.

Are we neglecting other techniques? The beginning of this Review opened with the principles, benefits and limitations of the traditional brain imaging techniques (PET and fMRI). Although these techniques have been incredibly revealing, and integration with other disciplines might help us reveal more, the limitations described earlier will invariably still remain. Thus, researchers and clinicians might be performing a disservice to themselves by continuing to overuse these techniques and not exploring alternatives. Table 1 highlights the principle features of alternative techniques, and notes key studies that have used these techniques in the hope that their use is encouraged in future brain imaging studies. As these techniques are still subject to certain limitations, there use might best serve in a multimodal context (alongside one another or with conventional brain imaging techniques), in that a potential limitation of one technique is overcome by the advantage of another. Interestingly, the lack of an organic biomarker or an identifiable endophenotype has always been a caveat in FGIDs. Studies using structural imaging approaches have, however, identified microstructural changes in patients, suggesting that FGIDs could have an organic component.95–97 Although the causality is unclear at present, these approaches might nevertheless allow us to better define biomarkers or endophenotypes, not only in gastroenterology, but also across other fields.

Conclusions

Our understanding of the human brain–gut axis has expanded exponentially over the past two decades.

Although this new growth has been incredibly revealing in some areas, in other areas the field has been plagued by a number of conflicting results, which has obscured our perspective of the underlying connection between the gut and the human brain and perhaps taken us ‘off track’, as researchers try to repeat experiments to clarify inconsistencies. The latter has, unfortunately, been the case for some brain imaging studies in gastroentero­ logy, and is the result of questionable methodological choices, unsubstantiated inferences and limited brain imaging modalities. Fortunately, some of these limitations of the field have been addressed; however, other areas are still in need of improvement. In summary, one course of action that is required is the integration of research from affective, cognitive and psychiatric neuro­s ciences, as well as behavioural and epidemio­ logical research that have all been shown to affect cerebral responses to gastro­intestinal responses. Researchers should integrate these aspects into the design of future studies utilizing brain-imaging resources, and needs to be applied at all stages: in study design, data collection and analysis. Moreover, researchers need to be aware of the limitations of brain imaging studies (some of which have been highlighted in this Review), so that results can be obtained that best relate to and reflect clinical situations. Furthermore, researchers need to be careful with the inferences they make. To address a specific question they should identify the most appropriate factors, including, among others, the correct brain-imaging analysis methods, MVPA and connectivity analyses. Finally, other novel neuroimaging methodology might need to be used to provide a different dimension of the brain–gut interaction, and whilst each methodological technique can have its limitations, collective utilization of different imaging modalities might inherently reduce limitations and provide improved understanding of brain process involved in a particular condition. Thus, standardization of gut stimulation methods, brain imaging parameters and selection of carefully phenotyped patients is imperative if the field is to advance. In conclusion, by adopting an overall integrative approach (Box 4), the use of brain imaging in gastrointestinal health and disease should get back on track, enabling identification of psychophysiological vulnerability factors in individuals and helping to identify biomarkers and endophenotypes that relate to visceral disorders. These approaches could provide the crucial first steps towards developing patient-tailored treatments, with improved efficacy, and might thus improve the quality of life of many.

Review criteria A literature search was performed in PubMed of articles published from 1946 until September 2013. The search terms used were “neuroimaging”, “gastrointestinal”, “visceral”, “pain”, “biomarker” and “FGID”, alone and in combination. All articles identified were English-language, full-text papers. We also searched the reference lists of identified articles for further relevant papers.

NATURE REVIEWS | GASTROENTEROLOGY & HEPATOLOGY © 2014 Macmillan Publishers Limited. All rights reserved

VOLUME 11  |  SEPTEMBER 2014  |  573

REVIEWS 1. 2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17. 18.

19.

20.

21.

22.

Jones, E. G. & Mendell, L. M. Assessing the decade of the brain. Science 284, 739 (1999). Aziz, Q. & Thompson, D. G. Brain-gut axis in health and disease. Gastroenterology 114, 559–578 (1998). Drossman, D. A. The functional gastrointestinal disorders and the Rome III process. Gastroenterology 130, 1377–1390 (2006). Van Oudenhove, L. & Aziz, Q. Recent insights on central processing and psychological processes in functional gastrointestinal disorders. Dig. Liver Dis. 41, 781–787 (2009). Talley, N. J. Functional gastrointestinal disorders as a public health problem. Neurogastroenterol. Motil. 20 (Suppl. 1), 121–129 (2008). Hongo, M. Epidemiology of FGID symptoms in Japanese general population with reference to life style. J. Gastroenterol. Hepatol. 26 (Suppl 3), 19–22 (2011). Boyce, P. M., Talley, N. J., Burke, C. & Koloski, N. A. Epidemiology of the functional gastrointestinal disorders diagnosed according to Rome II criteria: an Australian population-based study. Intern. Med. J. 36, 28–36 (2006). Derbyshire, S. W. A systematic review of neuroimaging data during visceral stimulation. Am. J. Gastroenterol. 98, 12–20 (2003). Mayer, E. A. et al. Brain imaging approaches to the study of functional GI disorders: a Rome working team report. Neurogastroenterol. Motil. 21, 579–596 (2009). Kupfer, D. J., Frank, E. & Phillips, M. L. Major depressive disorder: new clinical, neurobiological, and treatment perspectives. Lancet 379, 1045–1055 (2012). Phillips, M. L. & Kupfer, D. J. Bipolar disorder diagnosis: challenges and future directions. Lancet 381, 1663–1671 (2013). Pitman, R. K. et al. Biological studies of posttraumatic stress disorder. Nat. Rev. Neurosci. 13, 769–787 (2012). Dabbouseh, N. M. & Jensen, D. M. Future therapies for chronic hepatitis C. Nat. Rev. Gastroenterol. Hepatol. 10, 268–276 (2013). US Department of Health & Human services. The BRAIN initiative. Brain Research through Advancing Innovative Neurotechnologies (BRAIN). NIH [online], http://www.nih.gov/ science/brain/ (2013). Human Brain Project. The Human Brain Project [online], https://www.humanbrainproject.eu/ (2013). Hobson, A. R. & Aziz, Q. Brain imaging and functional gastrointestinal disorders: has it helped our understanding? Gut 53, 1198–1206 (2004). Craig, A. D. A new view of pain as a homeostatic emotion. Trends Neurosci. 26, 303–307 (2003). Apkarian, A. V., Bushnell, M. C., Treede, R. D. & Zubieta, J. K. Human brain mechanisms of pain perception and regulation in health and disease. Eur. J. Pain 9, 463–484 (2005). Tracey, I. & Mantyh, P. W. The cerebral signature for pain perception and its modulation. Neuron 55, 377–391 (2007). Tillisch, K. et al. Sex specific alterations in autonomic function among patients with irritable bowel syndrome. Gut 54, 1396–1401 (2005). Mayer, E. A. et al. Differences in brain responses to visceral pain between patients with irritable bowel syndrome and ulcerative colitis. Pain 115, 398–409 (2005). Berman, S. M. et al. Reduced brainstem inhibition during anticipated pelvic visceral pain correlates with enhanced brain response to the visceral stimulus in women with irritable bowel syndrome. J. Neurosci. 28, 349–359 (2008).

574  |  SEPTEMBER 2014  |  VOLUME 11

23. Downar, J., Mikulis, D. J. & Davis, K. D. Neural correlates of the prolonged salience of painful stimulation. Neuroimage 20, 1540–1551 (2003). 24. Legrain, V., Iannetti, G. D., Plaghki, L. & Mouraux, A. The pain matrix reloaded: a salience detection system for the body. Prog. Neurobiol. 93, 111–124 (2011). 25. Lee, M. C., Mouraux, A. & Iannetti, G. D. Characterizing the cortical activity through which pain emerges from nociception. J. Neurosci. 29, 7909–7916 (2009). 26. Strigo, I. A., Bushnell, M. C., Boivin, M. & Duncan, G. H. Psychophysical analysis of visceral and cutaneous pain in human subjects. Pain 97, 235–246 (2002). 27. Strigo, I. A., Duncan, G. H., Boivin, M. & Bushnell, M. C. Differentiation of visceral and cutaneous pain in the human brain. J. Neurophysiol. 89, 3294–3303 (2003). 28. Strigo, I. A., Albanese, M. C., Bushnell, M. C. & Duncan, G. H. Visceral and cutaneous pain representation in parasylvian cortex. Neurosci. Lett. 384, 54–59 (2005). 29. Hobday, D. I. et al. A study of the cortical processing of ano-rectal sensation using functional MRI. Brain 124, 361–368 (2001). 30. Dunckley, P. et al. A comparison of visceral and somatic pain processing in the human brainstem using functional magnetic resonance imaging. J. Neurosci. 25, 7333–7341 (2005). 31. Hoeger Bement, M., Weyer, A., Keller, M., Harkins, A. L. & Hunter, S. K. Anxiety and stress can predict pain perception following a cognitive stress. Physiol. Behav. 101, 87–92 (2010). 32. Vassend, O., Roysamb, E. & Nielsen, C. S. Five‑factor personality traits and pain sensitivity: a twin study. Pain 154, 722–728 (2013). 33. Zelman, D. C., Howland, E. W., Nichols, S. N. & Cleeland, C. S. The effects of induced mood on laboratory pain. Pain 46, 105–111 (1991). 34. Weisenberg, M. Cognitive aspects of pain and pain control. Int. J. Clin. Exp. Hypn. 46, 44–61 (1998). 35. Meagher, M. W., Arnau, R. C. & Rhudy, J. L. Pain and emotion: effects of affective picture modulation. Psychosom. Med. 63, 79–90 (2001). 36. Pezawas, L. et al. 5‑HTTLPR polymorphism impacts human cingulate-amygdala interactions: a genetic susceptibility mechanism for depression. Nat. Neurosci. 8, 828–834 (2005). 37. Stein, J. L. et al. A validated network of effective amygdala connectivity. Neuroimage 36, 736–745 (2007). 38. Labus, J. S. et al. Sex differences in brain activity during aversive visceral stimulation and its expectation in patients with chronic abdominal pain: a network analysis. Neuroimage 41, 1032–1043 (2008). 39. Phillips, M. L. et al. The effect of negative emotional context on neural and behavioural responses to oesophageal stimulation. Brain 126, 669–684 (2003). 40. Coen, S. J. et al. Negative mood affects brain processing of visceral sensation. Gastroenterology 137, 253–261.e1–e2 (2009). 41. Kim, H. Y., Akbar, M., Lau, A. & Edsall, L. Inhibition of neuronal apoptosis by docosahexaenoic acid (22:6n‑3). Role of phosphatidylserine in antiapoptotic effect. J. Biol. Chem. 275, 35215–35223 (2000). 42. Johnston, N. E., Atlas, L. Y. & Wager, T. D. Opposing effects of expectancy and somatic focus on pain. PLoS ONE 7, e38854 (2012). 43. Whitehead, W. E. & Palsson, O. S. Is rectal pain sensitivity a biological marker for irritable bowel



44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

syndrome: psychological influences on pain perception. Gastroenterology 115, 1263–1271 (1998). Gregory, L. J. et al. Cognitive modulation of the cerebral processing of human oesophageal sensation using functional magnetic resonance imaging. Gut 52, 1671–1677 (2003). Coen, S. J. et al. Effects of attention on visceral stimulus intensity encoding in the male human brain. Gastroenterology 135, 2065–2074 (2008). Silverman, D. H. et al. Regional cerebral activity in normal and pathological perception of visceral pain. Gastroenterology 112, 64–72 (1997). Wilder-Smith, C. H., Schindler, D., Lovblad, K., Redmond, S. M. & Nirkko, A. Brain functional magnetic resonance imaging of rectal pain and activation of endogenous inhibitory mechanisms in irritable bowel syndrome patient subgroups and healthy controls. Gut 53, 1595–1601 (2004). Song, G. H. et al. Cortical effects of anticipation and endogenous modulation of visceral pain assessed by functional brain MRI in irritable bowel syndrome patients and healthy controls. Pain 126, 79–90 (2006). Bishop, S. J. Trait anxiety and impoverished prefrontal control of attention. Nat. Neurosci. 12, 92–98 (2009). Naliboff, B. D. et al. Cerebral activation in patients with irritable bowel syndrome and control subjects during rectosigmoid stimulation. Psychosom. Med. 63, 365–375 (2001). Verne, G. N. et al. Central representation of visceral and cutaneous hypersensitivity in the irritable bowel syndrome. Pain 103, 99–110 (2003). Ringel, Y. et al. Regional brain activation in response to rectal distension in patients with irritable bowel syndrome and the effect of a history of abuse. Dig. Dis. Sci. 48, 1774–1781 (2003). Kwan, C. L. et al. Abnormal forebrain activity in functional bowel disorder patients with chronic pain. Neurology 65, 1268–1277 (2005). Naliboff, B. D. et al. Longitudinal change in perceptual and brain activation response to visceral. Gastroenterology 131, 352–365 (2006). Lu, C. L. & Chang, F. Y. Placebo effect in patients with irritable bowel syndrome. J. Gastroenterol. Hepatol. 26 (Suppl 3.), 116–118 (2011). Tillisch, K., Mayer, E. A. & Labus, J. S. Quantitative meta-analysis identifies brain regions activated during rectal distension in irritable bowel syndrome. Gastroenterology 140, 91–100 (2011). Lawal, A., Kern, M., Sidhu, H., Hofmann, C. & Shaker, R. Novel evidence for hypersensitivity of visceral sensory neural circuitry in. Gastroenterology 130, 26–33 (2006). Naliboff, B. D. & Mayer, E. A. Brain imaging in IBS: drawing the line between cognitive and non‑cognitive processes. Gastroenterology 130, 267–270 (2006). Berman, S. M. et al. Enhanced preattentive central nervous system reactivity in irritable bowel syndrome. Am. J. Gastroenterol. 97, 2791–2797 (2002). Ploghaus, A., Becerra, L., Borras, C. & Borsook, D. Neural circuitry underlying pain modulation: expectation, hypnosis, placebo. Trends Cogn. Sci. 7, 197–200 (2003). Porro, C. A., Cettolo, V., Francescato, M. P. & Baraldi, P. Functional activity mapping of the mesial hemispheric wall during anticipation of pain. Neuroimage 19, 1738–1747 (2003).

www.nature.com/nrgastro © 2014 Macmillan Publishers Limited. All rights reserved

REVIEWS 62. Yaguez, L. et al. Brain response to visceral aversive conditioning: a functional magnetic resonance imaging study. Gastroenterology 128, 1819–1829 (2005). 63. Tillisch, K. et al. Consumption of fermented milk product with probiotic modulates brain activity. Gastroenterology 144, 1394–1401 (2013). 64. Jalanka-Tuovinen, J. et al. Intestinal microbiota in healthy adults: temporal analysis reveals individual and common core and relation to intestinal symptoms. PLoS ONE 6, e23035 (2011). 65. Aziz, Q., Dore, J., Emmanuel, A., Guarner, F. & Quigley, E. M. Gut microbiota and gastrointestinal health: current concepts and future directions. Neurogastroenterol. Motil. 25, 4–15 (2013). 66. European Cooperation in Science and Technology. The Genes in Irritable Bowel Syndrome Research Network Europe (GENIEUR). European Coooperation in Science and Technology [online], http://www.cost.eu/ domains_actions/bmbs/Actions/BM1106 (2014). 67. The Oppenheimer Center for Neurobiology of Sress at UCLA. Pain and interoception Imaging Network Repository [online], http:// pain.med.ucla.edu/ (2014). 68. Ramsey, J. D. et al. Six problems for causal inference from fMRI. Neuroimage 49, 1545–1558 (2010). 69. Poldrack, R. A. Can. cognitive processes be inferred from neuroimaging data? Trends Cogn. Sci. 10, 59–63 (2006). 70. Poldrack, R. A. et al. Guidelines for reporting an fMRI study. Neuroimage 40, 409–414 (2008). 71. Mouraux, A., Diukova, A., Lee, M. C., Wise, R. G. & Iannetti, G. D. A multisensory investigation of the functional significance of the “pain matrix”. Neuroimage 54, 2237–2249 (2011). 72. Mouraux, A. & Iannetti, G. D. Nociceptive laser‑evoked brain potentials do not reflect nociceptive-specific neural activity. J. Neurophysiol. 101, 3258–3269 (2009). 73. Iannetti, G. D., Salomons, T. V., Moayedi, M., Mouraux, A. & Davis, K. D. Beyond metaphor: contrasting mechanisms of social and physical pain. Trends Cogn. Sci. 17, 371–378 (2013). 74. Hutzler, F. Reverse inference is not a fallacy per se: Cognitive processes can be inferred from functional imaging data. Neuroimage 84, 1061–1069 (2013). 75. Haxby, J. V. Multivariate pattern analysis of fMRI: the early beginnings. Neuroimage 62, 852–855 (2012). 76. Liang, M., Mouraux, A., Hu, L. & Iannetti, G. D. Primary sensory cortices contain distinguishable spatial patterns of activity for each sense. Nat. Commun. 4, 1979 (2013). 77. Labus, J. S. et al. Brain networks underlying perceptual habituation to repeated aversive visceral stimuli in patients with irritable bowel syndrome. Neuroimage 47, 952–960 (2009). 78. Labus, J. S. et al. Acute tryptophan depletion alters the effective connectivity of emotional arousal circuitry during visceral stimuli in healthy women. Gut 60, 1196–1203 (2011). 79. Wagner, J. A. Biomarkers: principles, policies, and practice. Clin. Pharmacol. Ther. 86, 3–7 (2009). 80. Lathia, C. D. et al. The value, qualification, and regulatory use of surrogate end points in drug development. Clin. Pharmacol. Ther. 86, 32–43 (2009). 81. Tillisch, K. & Labus, J. S. Advances in imaging the brain-gut axis: functional gastrointestinal disorders. Gastroenterology 140, 407–411.e1 (2011).

82. Biswal, B. B. et al. Toward discovery science of human brain function. Proc. Natl Acad. Sci. USA 107, 4734–4739 (2010). 83. Gottesman, II & Gould, T. D. The endophenotype concept in psychiatry: etymology and strategic intentions. Am. J. Psychiatry 160, 636–645 (2003). 84. Greicius, M. Resting-state functional connectivity in neuropsychiatric disorders. Curr. Opin. Neurol. 21, 424–430 (2008). 85. Baliki, M. N., Geha, P. Y., Apkarian, A. V. & Chialvo, D. R. Beyond feeling: chronic pain hurts the brain, disrupting the default-mode network dynamics. J. Neurosci. 28, 1398–1403 (2008). 86. Hong, J. Y. et al. Patients with chronic visceral pain show sex-related alterations in intrinsic oscillations of the resting brain. J. Neurosci. 33, 11994–12002 (2013). 87. Farmer, A. D., Aziz, Q., Tack, J. & Van Oudenhove, L. The future of neuroscientific research in functional gastrointestinal disorders: integration towards multidimensional (visceral) pain endophenotypes? J. Psychosom. Res. 68, 475–481 (2010). 88. Coen, S. J. et al. Neuroticism influences brain activity during the experience of visceral pain. Gastroenterology 141, 909–917.e1 (2011). 89. Farmer, A. D. et al. Psychophysiological responses to pain identify reproducible human clusters. Pain 154, 2266–2276 (2013). 90. Kosek, E., Jensen, K. B., Lonsdorf, T. B., Schalling, M. & Ingvar, M. Genetic variation in the serotonin transporter gene (5-HTTLPR, rs25531) influences the analgesic response to the short acting opioid Remifentanil in humans. Mol. Pain 5, 37 (2009). 91. Lindstedt, F., Lonsdorf, T. B., Schalling, M., Kosek, E. & Ingvar, M. Perception of thermal pain and the thermal grill illusion is associated with polymorphisms in the serotonin transporter gene. PLoS ONE 6, e17752 (2011). 92. Palit, S. et al. Serotonin transporter gene (5-HTTLPR) polymorphisms are associated with emotional modulation of pain but not emotional modulation of spinal nociception. Biol. Psychol. 86, 360–369 (2011). 93. Farmer, A. D. et al. Psychophysiological responses to visceral and somatic pain in functional chest pain identify clinically relevant pain clusters. Neurogastroenterol. Motil. 26, 139–148 (2013). 94. Holschneider, D. P., Bradesi, S. & Mayer, E. A. The role of experimental models in developing new treatments for irritable bowel syndrome. Expert Rev. Gastroenterol. Hepatol. 5, 43–57 (2011). 95. Zhou, G. et al. White-matter microstructural changes in functional dyspepsia: a diffusion tensor imaging study. Am. J. Gastroenterol. 108, 260–269 (2013). 96. Mayer, E. A., Tillisch, K. & Ellingson, B. M. Dyspepsia: Structural changes in functional gastrointestinal disorders. Nat. Rev. Gastroenterol. Hepatol. 10, 200–202 (2013). 97. Ellingson, B. M. et al. Diffusion tensor imaging detects microstructural reorganization in the brain associated with chronic irritable bowel syndrome. Pain 154, 1528–1541 (2013). 98. Huettel, S. A., Song, A. W. & McCarthy, G. (Eds) Functional Magnetic Resonance Imaging 2nd Edition 159–184 (Sinauer Associates, 2008). 99. Schweinhardt, P., Bountra, C. & Tracey, I. Pharmacological FMRI in the development of new analgesic compounds. NMR Biomed. 19, 702–711 (2006). 100. Wise, R. G. & Preston, C. What is the value of human FMRI in CNS drug development? Drug Discov. Today 15, 973–980 (2010).

NATURE REVIEWS | GASTROENTEROLOGY & HEPATOLOGY © 2014 Macmillan Publishers Limited. All rights reserved

101. Logothetis, N. K., Pauls, J., Augath, M., Trinath, T. & Oeltermann, A. Neurophysiological investigation of the basis of the fMRI signal. Nature 412, 150–157 (2001). 102. Nagaoka, T. et al. Increases in oxygen consumption without cerebral blood volume change during visual stimulation under hypotension condition. J. Cereb. Blood Flow Metab. 26, 1043–1051 (2006). 103. Schachinger, H., Klarhofer, M., Linder, L., Drewe, J. & Scheffler, K. Angiotensin II decreases the renal MRI blood oxygenation level-dependent signal. Hypertension 47, 1062–1066 (2006). 104. Raichle, M. E. Behind the scenes of functional brain imaging: a historical and physiological perspective. Proc. Natl Acad. Sci. USA 95, 765–772 (1998). 105. Fox, P. T., Burton, H. & Raichle, M. E. Mapping human somatosensory cortex with positron emission tomography. J. Neurosurg. 67, 34–43 (1987). 106. Dolle, F. [18F]fluoropyridines: from conventional radiotracers to the labeling of macromolecules such as proteins and oligonucleotides. In Ernst Schering Research Foundation Workshop (Eds. Schubiger, P. A., Lehmann, L. & Friebe, M.) 113–157 (Springer, 2007). 107. Derbyshire, S. W. & Jones, A. K. Cerebral responses to a continual tonic pain stimulus measured using positron emission tomography. Pain 76, 127–135 (1998). 108. Henningsen, P., Zimmermann, T. & Sattel, H. Medically unexplained physical symptoms, anxiety, and depression: a meta-analytic review. Psychosom. Med. 65, 528–533 (2003). 109. Furlong, P. L. et al. Dissociating the spatiotemporal characteristics of cortical neuronal activity associated with human volitional swallowing in the healthy adult brain. Neuroimage 22, 1447–1455 (2004). 110. Worthen, S. F., Hobson, A. R., Hall, S. D., Aziz, Q. & Furlong, P. L. Primary and secondary somatosensory cortex responses to anticipation and pain: a magnetoencephalography study. Eur. J. Neurosci. 33, 946–959 (2011). 111. Smith, J. K. et al. fMRI and MEG analysis of visceral pain in healthy volunteers. Neurogastroenterol. Motil. 23, 648–e260 (2011). 112. Sami, S. A. et al. Cortical changes to experimental sensitization of the human esophagus. Neuroscience 140, 269–279 (2006). 113. Dimcevski, G. et al. Pain in chronic pancreatitis: the role of reorganization in the central nervous system. Gastroenterology 132, 1546–1556 (2007). 114. Olesen, S. S., Hansen, T. M., Graversen, C., Valeriani, M. & Drewes, A. M. Cerebral excitability is abnormal in patients with painful chronic pancreatitis. Eur. J. Pain 17, 46–54 (2013). 115. Detre, J. A., Rao, H., Wang, D. J., Chen, Y. F. & Wang, Z. Applications of arterial spin labeled MRI in the brain. J. Magn. Reson. Imaging 35, 1026–1037 (2012). 116. Detre, J. A., Wang, J., Wang, Z. & Rao, H. Arterial spin-labeled perfusion MRI in basic and clinical neuroscience. Curr. Opin. Neurol. 22, 348–355 (2009). 117. Kano, M. et al. Physiological and psychological individual differences influence resting brain function measured by ASL perfusion. Brain Struct. Funct. http://dx.doi.org/10.1007/ s00429-013-0593-8. 118. Owen, D. G., Clarke, C. F., Ganapathy, S., Prato, F. S. & St. Lawrence, K. S. Using perfusion MRI to measure the dynamic changes

VOLUME 11  |  SEPTEMBER 2014  |  575

REVIEWS in neural activation associated with tonic muscular pain. Pain 148, 375–386 (2010). 119. Mori, S. & Zhang, J. Principles of diffusion tensor imaging and its applications to basic neuroscience research. Neuron 51, 527–539 (2006). 120. Tsurugizawa, T., Ciobanu, L. & Le Bihan, D. Water diffusion in brain cortex closely tracks underlying neuronal activity. Proc. Natl Acad. Sci. USA 110, 11636–11641 (2013). 121. Blankstein, U., Chen, J., Diamant, N. E. & Davis, K. D. Altered brain structure in irritable bowel syndrome: potential

576  |  SEPTEMBER 2014  |  VOLUME 11

contributions of pre-existing and disease-driven factors. Gastroenterology 138, 1783–1789 (2010). 122. Labus, J. et al. Irritable bowel syndrome in female patients is associated with alterations in structural brain networks. Pain 155, 137–149 (2013). 123. Seminowicz, D. A. et al. Regional gray matter density changes in brains of patients with irritable bowel syndrome. Gastroenterology 139, 48–57 (2010). 124. Frokjaer, J. B. et al. Reduced cortical thickness of brain areas involved in pain processing



in patients with chronic pancreatitis. Clin. Gastroenterol. Hepatol. 10, 434–438.e1 (2012). 125. Apkarian, A. V. et al. Chronic back pain is associated with decreased prefrontal and thalamic gray matter density. J. Neurosci. 24, 10410–10415 (2004). Author contributions Y.A.O. researched data for and wrote the article. Both authors made substantial contributions to discussion of content and reviewing/editing the manuscript before submission.

www.nature.com/nrgastro © 2014 Macmillan Publishers Limited. All rights reserved