Enlightening the frontiers of neurogastroenterology through optogenetics

Abstract

Neurogastroenterology refers to the study of the extrinsic and intrinsic nervous system circuits controlling the gastrointestinal (GI) tract. Over the past 5–10 yr there has been an explosion in novel methodologies, technologies and approaches that offer great promise to advance our understanding of the basic mechanisms underlying GI function in health and disease. This review focuses on the use of optogenetics combined with electrophysiology in the field of neurogastroenterology. We discuss how these technologies and tools are currently being used to explore the brain-gut axis and debate the future research potential and limitations of these techniques. Taken together, we consider that the use of these technologies will enable researchers to answer important questions in neurogastroenterology through fundamental research. The answers to those questions will shorten the path from basic discovery to new treatments for patient populations with disorders of the brain-gut axis affecting the GI tract such as irritable bowel syndrome (IBS), functional dyspepsia, achalasia, and delayed gastric emptying.

INTRODUCTION

The term “neurogastroenterology” was coined in the late 1990s to encompass the interdisciplinary research demonstrating the importance of the central and peripheral nervous systems in the control of the gastrointestinal (GI) tract (110). The brain and spinal cord provide the extrinsic nerves that innervate the GI tract, whereas the intrinsic neural innervation is provided by the enteric nervous system (ENS). Despite great progress in the field of neurogastroenterology over recent years with identifying physiological mechanisms for disorders such as irritable bowel syndrome (IBS), functional dyspepsia, and gastroparesis (47, 90, 102), we consider that the underlying mechanisms by which the central nervous system (CNS) regulates gut function requires further investigation. In this review we explore the latest optogenetic tools, which are being used in real-time to dissect the neural circuits regulating visceral nociception and GI motility in freely moving animals. In addition, we provide an overview of how electrophysiological methods can be combined with optogenetics to further expand our understanding of supraspinal, spinal, and enteric mechanisms that control the gut. Future interventions using optogenetic and electrophysiological methods may prove pivotal in the search for novel approaches in the management of GI dysmotility and visceral pain (Fig. 1). We believe that readers of this review will gain an advanced understanding of optogenetics that will enable them to ask key research questions that have been difficult to answer with older techniques, such as: how do specific neuronal populations modulate GI sensitivity or motility?; or can specific brain, spinal, or enteric circuits be manipulated to change GI function? Optogenetics, in conjunction with other experimental paradigms, can be used to address those questions by directly manipulating neurons to change function or behavior, which in turn, will shorten the path from basic discovery to new treatments for patients with disorders of the GI tract such as IBS, functional dyspepsia, achalasia, and delayed gastric emptying.

Fig. 1.

Optogenetics and electrophysiology in neurogastroenterology. Within the next decade optogenetic and electrophysiological tools will greatly advance the field of neurogastroenterology. Specific targeting within the enteric nervous system (ENS) and the central nervous system (CNS; brain and spinal cord), along with light delivery strategies that are being developed, will allow the use of optogenetics to directly change gastrointestinal (GI) motility and sensitivity to actively reverse pathological process. Complementing the optogenetic tools, additional electrophysiological approaches are being developed to monitor abnormal motility and sensitivity within the GI tract and/or integrated signaling within the CNS. Real-time measurement of the electrical processes will permit closed-loop systems with optogenetic approaches to modify GI functions and other electrophysiological tools will be used to identify key neurotransmitters and ion channels underlying GI pathophysiology.

OPTOGENETICS

Optogenetics refers to using genetic manipulation and light stimulation to achieve precise temporal control of processes in living cells (37, 111). The most common application of optogenetics has been to induce or inhibit action potentials in discrete neurons, resulting in changes in neurotransmitter release, which may result in changing the behavior of the freely moving animal. When stimulating light-sensitive channels expressed in specific neuronal circuits, such as channelrhodopsin (ChR), a sodium channel, or halorhodopsin (HR), a chloride pump, real-time changes in animal behavior could be evoked (44, 113). The current use of optogenetic techniques to control neuronal activity was developed in the mid-2000s by Karl Deisseroth’s group at Stanford (17). Studies by the Deisseroth laboratory, Tye laboratory, as well as many others have used optogenetic tools, including transgenic or virally mediated opsins, to investigate brain circuits that modify different behaviors such as anxiety-like behavior, depression-like behavior, fear, memory, and nociception (9, 13, 20, 72). Optogenetic tools have also been used in the spinal cord, dorsal root ganglia (DRG) cell bodies, and peripheral nerves to study the neuronal pathways underlying nociception (26, 54, 75). Other optogenetic tools target G protein-coupled signaling pathways for studying the effects of intracellular signaling on cellular function (37, 111). Finally, an additional definition of optogenetics in the field of neurogastroenterology is applied to the use of genetically encoded calcium indicators based on the enhanced green fluorescent protein-calmodulin-M13 fragment of the myosin light chain kinase fusion protein (GCaMP) for endogenous tracking of intracellular calcium (70).

USE OF OPTOGENETICS IN THE ENS TO MANIPULATE GI FUNCTION

After a decade from the initial publications using optogenetics in neuroscience, the field of neurogastroenterology has begun to apply optogenetic tools to both in vivo and ex vivo preparations to investigate peripheral and central circuits modifying GI function along the brain-gut axis. In the first proof-of-principal study to target the ENS in mice, Stamp et al. (98) transplanted enteric neural stem cells expressing ChR into the colon of healthy recipient mice. They found that the transplanted cells matured into fully functional ENS nerve networks containing excitatory and inhibitory motor neurons as well as interneurons capable of changing contractility in an organ bath following light-induced activation of ChR (98). While this study did not demonstrate cell-type specificity since three classes of neurons developed from the stem cells, the use of ChR ensured that only activation of the transplanted cells produced the change in function, providing support for the usefulness of cell-based therapies for patients with enteric neuropathies. Mice with ChR expressed only in cholinergic neurons were used to selectively characterize excitatory and inhibitory junction potentials in smooth muscle cells evoked by light stimulation or electrical stimulation along with application of selective antagonists to characterize the classes of neurotransmitters released by stimulation (77). This study highlighted the advantage of selective optogenetic stimulation to identify cell-type specific responses by directly comparing changes in junction potentials between light and electrical stimulation. Using a different genetic approach, restricting ChR expression to muscularis macrophages was sufficient to cause light-induced colonic smooth muscle contractions, mediated by prostaglandin E2, in an organ bath, demonstrating a non-neuronal component to colonic motility that could not have been demonstrated without the selective optogenetic activation (62). In another study, Hibbard et al. (50) used transgenic mice in which ChR was targeted to calretinin positive (predominantly cholinergic) neurons and found that light stimulation of the proximal colon was able to induce propulsive motility both in vivo, using a wireless LED, and in organ bath preparations, thus providing evidence that selective expression of ChR in the ENS could be sufficient to induce changes in GI motility. In transgenic mice with ChR expression limited to nitrergic enteric neurons, light stimulation of those cells inhibited the propagation of colonic migrating motor complexes measured in organ baths, with a resumption of the motility pattern once the stimulation stopped (43), findings that were supported by motility-induced changes in calcium transients from nitrergic or cholinergic nerves in the ENS expressing GCaMP. In mice with ChR restricted to colonic epithelial cells, organ bath studies demonstrated that ChR stimulation was sufficient to activate different classes of extrinsic afferents, while light stimulation within the lumen of the colon in conscious mice was sufficient to elicit a visceromotor response that was likely mediated by purinergic signaling, providing direct evidence of epithelial to neural communication (64). Finally, taking advantage of naturally transparent Zebra fish larva, ChR was expressed on hyperpolarization-activated cyclic nucleotide-gated type 4 expressing serotonergic cells in the GI tract and stimulation of these cells enhanced retrograde peristalsis, suggesting a possible role for these cells in the local regulation of emetic motility patterns (40). Thus, these diverse studies have advanced the field of neurogastroenterology through the use of optogenetic strategies to evaluate how specific cells within the gut modulate GI function, which could lead to new therapies for GI motility or sensory disorders.

Studies using GCaMP have also added to the field’s understanding of GI physiology. In particular, the Sanders’ laboratory (6, 7, 30–33) has generated a murine model that expresses the GCaMP in interstitial cells of Cajal (ICC) and have used ex vivo preparations to study mechanisms of pacemaker activity and slow wave generation in a variety of GI tissues. Similarly, the Gulbransen group (27, 28) has used mice with GCaMP expressed in enteric glia to demonstrate their role in regulating GI motility as well as their participation in tachykinin mediated neuroinflammation. Finally, complementary studies from the Vanden Berghe laboratory (15, 61) used transgenic mice that expressed GCaMP in enteric nerves and enteric glia to identify pannexin channels as mediators of neural-glia communication and to demonstrate that colonic motility patterns are ‘hardwired’ within enteric nerves, with regional differences in the complexity of the possible motility patterns. As such, these optogenetic-adjacent tools to monitor intracellular calcium have provided novel insights into how motility occurs within the GI tract that would have been difficult to determine using other techniques.

USE OF OPTOGENETICS IN THE CNS TO MANIPULATE GI FUNCTION

The first published studies of optogenetics using opsins in the field of neurogastroenterology used mice that expressed ChR on sensory nerves to demonstrate that, in an organ bath, specific extrinsic primary colorectal afferents could be stimulated by the focal application of light trains (116). Furthermore, those same type of transgenic mice were used to characterize mechanically insensitive afferents that could be sensitized following a combination of light-stimulation and algesic chemical exposure (36). In more recent studies from the Davis laboratory (64), light stimulation within the lumen of the colon in mice with ChR expressed in afferents expressing transient receptor potential cation channel subfamily V member 1 directly elicited a visceromotor response. In a follow-up study, the same group demonstrated that the light-evoked visceromotor response induced cFos activation within the dorsal horn of the spinal cord, and that activation of extrinsic afferents led to changes in colonic contractility via a spinally mediated modulation of enteric nerves (92). In our laboratory, we demonstrated a direct role for limbic circuitry to modify colonic sensitivity in the rat by stimulating terminals containing virally expressed ChR or HR from the central nucleus of the amygdala (CeA) at the bed nucleus of the stria terminalis (BNST). Visceral hypersensitivity to colonic distension in normal rats could be evoked by ChR stimulation of the CeA-BNST pathway, whereas HR inhibition was without effect (56). Thus, these studies demonstrated that both “top-down” and “bottom-up” manipulation of colonic sensitivity was achieved with optogenetic techniques providing support for the potential of these tools to modify visceral sensitivity in functional GI disorders. To classify the extrinsic neurons that innervate the GI tract, techniques have been developed independently by the Powley and Spencer laboratories (59, 80, 81, 96) to selectively label extrinsic afferents that innervate the GI tract by targeting the cell bodies in the DRG for spinal afferents or the nodose ganglia for vagal afferents. Specifically labeling afferent nerves with endings within the GI tract cannot only be used to selectively study neuronal properties and distribution, but also presents a strategy to target these nerves for optogenetic manipulation. Appling these labeling strategies, Han et. al. (46) achieved specific targeting of vagal afferents by using a combination of virally mediated retrograde Cre expression from the gut and Cre-mediated ChR expression at the nodose ganglia. The group then used light stimulation of nodose ganglia fibers at the nucleus tractus solitarius (NTS) or the area postrema to demonstrated functional roles for those afferents. The authors found that the right nodose ganglia mediated reward behaviors via an NTS-parabrachial nucleus-substantia nigra (SN) pathway that caused dopamine release, while the left nodose ganglia mediated satiety signals via the area postrema (46). For vagal efferents, Anselmi et. al. used virally mediated expression of HR in the SN of rats with light-induced inhibition of terminals from the SN at the dorsal motor nucleus of the vagus, which directly reduced gastric smooth muscle tone and contractility (3). Furthermore, using transgenic mice expressing ChR on GABAergic somatostatin neurons in the NTS in combination with photostimulation and pharmacology, a parasympathetic circuit that could regulate vagal control of gastric motility was demonstrated in a brain-slice preparation (60). In an in vivo preparation, by crossing Cre mouse lines to allow for selective expression of ChR, separate roles in modulating GI function for two distinct vagal neuronal populations expressing either the orphan purinergic receptor GPR65 or the glucagon-like peptide 1 receptor (GLP1R) were demonstrated. Specifically, GPR65 expressing afferents could detect nutrients, and ChR activation decreased gastric pressure and inhibited gastric motility; in contrast, GLP1R expressing afferents detected stretch and ChR activation increased gastric pressure and motility (104). More recently, a study crossed several Cre mouse lines selective for neuronal subtypes, such as GPR65, GLP1R, vasoactive intestinal peptide, or oxytocin receptor, and others, to visualize and characterize receptor expression of vagal afferent and efferent neurons at the single-cell level. Once the phenotypes were molecularly and anatomically characterized, neuronal sub-type specific ChR expression was achieved by infecting the nodose ganglia, and light stimulation at the NTS was used to determine the functional role of the neuronal subtypes in feeding and water ingestion. The authors found that ChR stimulation of either GLP1R or oxytocin receptor expressing afferents decreased fasting-induced feeding without effect on thirst-induced water consumption, whereas the afferents expressing other markers did not change the food or water intake (5). The same group also found that within the hypothalamus, selective expression and stimulation of ChR in agouti-related peptide positive neurons demonstrated a leptin-independent increase in fasting-induced feeding behavior in mice, indicating that not only brainstem, but also central brain circuitry can be manipulated to change GI function (11). Thus, taking advantage of cell-specific ChR expression, the authors of these studies were able to apply optogenetics to identify CNS circuits regulating GI motility as well as integrated behaviors such as hunger and reward. In summary, the use of optogenetic tools in neurogastroenterology has significantly advanced our understanding of specific mediators of GI motility and sensitivity but many questions still remain to be addressed, such as: can we identify novel circuits or novel neurotransmitters within those circuits that influence GI function? how are CNS and/or ENS circuits changed in chronic disease?; or can altered motility and/or sensitivity be restored to normal function through selective excitation or inhibition of neurons in the CNS or ENS? Within a decade, we should have answers to those and other questions about GI physiology.

ELECTROPHYSIOLOGY

Electrophysiology is a broad term that at its core is the study of mechanisms that cause a change in electrical potential across a cell membrane. In the context of neurogastroenterology, electrophysiology is the study of the electrical properties of tissues throughout the GI tract and the nerves of the CNS that affect GI physiology. The studies can either be at the cellular level where changes in ion channels determine the effect on membrane excitability or at the level of the tissue or organ to determine the net effect on physiology. Electrophysiological recordings have two distinct, but related, applications for the field of neurogastroenterology. The “neuro-” component measures electrical properties of intrinsic neurons in the ENS, as well as the supporting glia, and how they influence motility, secretion, and absorption, or how extrinsic nerves, and glia, in the CNS participate in sensation and central reflexes that modulate GI function. The “-gastroenterology” component measures electrical properties of other tissues within the GI tract, such as smooth muscle, epithelium, enteroendocrine cells, and ICC. For instance, the GI mucosa is composed of a polarized epithelium with cell membranes that permit selective transport of both charged and uncharged molecules of various sizes either through or between cells, whereas smooth muscles are innervated by nerves and ICC cells to modulate contractility (86). On the application side, electrophysiology can also refer to techniques that use electrical stimulation to affect GI functions as well as monitoring motility throughout the GI tract (18, 34, 38, 74).

Electrophysiology in gastroenterology has been used to identify changes in electrical events within the GI tract, in animal or human tissues, to improve our understanding of the underlying mechanisms of visceral sensitivity or motility (79, 107, 108). For example, a recent study of colonic motility in the guinea pig from the Spencer laboratory (24) combined video recordings of tissue movement, pressure manometry, and smooth muscle electrophysiology to characterize three different motility patterns underlying fecal pellet output. The same team was also able to image neuronal calcium transients simultaneously with smooth muscle contractility to demonstrate how ENS depolarizations could lead to motility in an ex vivo mouse colon preparation (95). For studying electrophysiological properties of the GI tract, one can start with the luminal surface from different regions of the GI tract, tissue culture systems, or clinical biopsies. The electrical properties of that surface epithelia can be measured in Ussing chambers to assess changes in ion channels and/or tight junctions that affect permeability (19, 22, 100). Selective pharmacological tools can be applied to the chambers to determine the effect of different classes of receptors on ion transport, and electrical field stimulation can be used to directly activate voltage-gated channels or nerve terminals within the tissues. Similar studies can be conducted in muscle baths using force transducers to measure changes in contractility of individual smooth muscle layers or full thickness tissue segments. In more complex ex vivo systems, sheets of smooth muscle can be prepared that allow for directly measuring electrical properties of ENS nerves in the myenteric plexus, and extrinsic afferent recordings can be conducted by stimulating the organ in the bath and recording from the afferent terminals (35). Calcium imaging can also be used to directly monitor neuronal and glial activity in the ENS (39). Recently, organ bath techniques have been applied to the first study of human visceral nociceptors from GI tissues obtained following surgeries in which mechanical and sensory responses of the fibers were characterized (66). Reverse translational studies have also been performed in which several sodium channelopathies identified from genotype studies of IBS patients were exogenously expressed in cell culture allowing for the characterization of the biophysical properties of the mutant channels (99). Overall, scientists have used a combination of electrophysiological techniques, including afferent fiber recordings, single-unit recordings, multi-electrode arrays, field potentials, patch clamping, voltage-sensitive dyes, and calcium reporters to monitor properties of enteric neurons (45, 48, 82, 91), enteric glia (16, 45, 48), ICC (87), DRG (35, 106), spinal neurons (85, 89, 103), spinal glia, and neurons (21, 78) and glia (14, 25, 57) throughout the brain to determine underlying pathophysiological mechanisms of GI disorders.

What are some of the new state-of-the-art techniques in GI electrophysiology? The Nakayama group has developed a microelectrode array lined with a dialysis membrane to study pacemaker activity, local neurotransmission, and oscillating spike potentials with greater resolution than optical probes in murine tissues (53, 69). Nishimura et al. (73) imaged changes in calcium transients in the ENS with simultaneous recordings of cortical field potentials evoked by luminal stimuli to demonstrate a direct gut-brain connection. In clinical studies, while diagnostic motility studies rely on manometry or impedance measurements, electrophysiological techniques have also been used to measure motility patterns in different GI organs (23, 101). In proof-of-concept preclinical studies in pigs, optical imaging techniques using voltage sensitive dyes were used to monitor gastric slow waves with results that were comparable to more traditional electrical recording techniques (114). Gastric motility could also be measured using an electrode applied noninvasively to the mucosal surface of the pig’s stomach with a resolution that was comparable to surgically implanted electrodes (1). More recently, direct measurement of gastric and small intestine motility in humans has been conducted using flexible, high resolution multi-electrode arrays applied acutely during surgery (2, 10), while rectal and colonic afferent nerve activity from clinical samples has been measured in organ baths (71, 76). Those studies provide direct information about human motility patterns and neurophysiology that can be applied to future studies in patients with GI disorders and will permit researchers to compare findings from animal models to bolster preclinical studies for new therapeutics. Because of the multiple methods and tissues, an exhaustive description of electrophysiological techniques that can be used in neurogastroenterology is beyond the scope of this review; nonetheless, the authors refer the readers to other focused reviews for further detailed reading (12, 29, 49, 52, 87, 109).

FUTURE APPLICATIONS OF OPTOGENETIC IN COMBINATION WITH ELECTROPHYSIOLOGICAL TOOLS TO ADVANCE NEUROGASTROENTEROLOGY

Currently, optogenetics and electrophysiology are complementary, but separate, technologies, depending on the question being asked. While electrophysiology can be used to verify opsin activity by measuring evoked changes in electrical activity, it can also be an independent tool for modulating or monitoring GI functions, with less specificity than optogenetic manipulations. Looking forward, there are exciting possibilities for the use of integrated optogenetic and electrophysiological tools for investigating and/or treating GI disorders. While initial optogenetic studies required tethering to fiber optic cables for light delivery, wireless options for light delivery have been recently developed (55, 65, 115). These advances will permit the unobstructed study of behavioral or functional changes in response to manipulation of the affected system. Recent reviews provide more detailed discussion about the potential for wireless optogenetic manipulation of GI sensation and motility (93, 94), with the goal of developing clinically applicable technologies to treat GI disorders. There are also many opsins with different channel properties for circuit excitation or inhibition that can be used in addition to ChR or HR. In particular, step-function opsins avoid the potential of off-target heating effects caused by prolonged light stimulation in that they are activated by a brief exposure of one wavelength of light but remain active until deactivated by a second, different wavelength. ChR has also been mutated to increase the kinetics of the channel to allow for high-frequency stimulation, red-shifted to permit activation by lower energy light that can penetrate deeper into tissues, and changed from a sodium to a chloride channel to mediate inhibition rather than excitation (58, 97). Optogenetic tools are also being applied to clustered regularly interspaced short palindromic repeat technologies to modify gene expression. By fusing light-sensitive transcription factors to an inactivated Cas9 nuclease and targeting with an appropriate short-guide RNA, gene expression can be directly enhanced following light stimulation, independent of endogenous activators or repressors (41). Neurogastroenterologists will be able to answer really tough questions in our field. For example, with careful targeting of optogenetic constructs to specific cellular populations, they will be able to investigate how activation, inhibition, or other modulation of those cells affects GI physiology. Once basic questions are answered, studies in disease models can then identify pathophysiological mechanisms, thus providing opportunities for novel therapeutic interventions. For electrophysiological tools, closed-loop electrical stimulation devices are being used to treat epilepsy, deep brain stimulation is being assessed for efficacy to treat depression, spinal cord stimulators are used for inhibiting pain, and pacing electrodes are being evaluated for correcting GI motility disorders (67, 68, 88). Additionally, flexible microelectrodes are currently in development that can accommodate chronic implantation and monitoring of the target organ (4, 84), including the ENS (8). Thus, one could envision a combined optogenetic and electrophysiological approach where a self-contained electrophysiological monitoring system detects abnormal motility or sensory neuronal activity and responds by triggering light stimulation to restore the targeted function. Another future approach could be to modify smart-pill technology to emit light to directly stimulate enteric nerves expressing opsins to modify motility or secretion.

CHALLENGES AND LIMITATIONS

The first significant challenge of optogenetics in combination with electrophysiology is access to the target tissue. While the esophagus, stomach, duodenum, and colon are relatively easy to access for gastroenterologists, targeting DRG, the spinal cord, or the brain generally requires surgical interventions. Indeed, complex surgical techniques have been developed in recent years to directly access DRG in mice as survival surgery, which means that neuronal tracers or viruses and micro-LEDs can be implanted directly at these sites (96). In animal models and in the clinic, stereotaxic techniques are used for minimally invasive targeting of discrete areas of the CNS. Noninvasive monitoring options are limited to cortical brain structures near the surface of the head or cranial nerves, such as the vagus. Spectrum-shifted opsins could be used that respond to near-infrared light that has better tissue penetration to permit the light source to be placed superficially or externally, with the limitation that deep CNS structures could not be targeted (112). Borrowing again from the epilepsy literature, the amygdala and hippocampus can be targeted with magnetic resonance imaging guidance for laser ablation treatment, suggesting that limbic areas could be targeted for optogenetic manipulation in a similar manner (105).

The second significant challenge for optogenetics is the need to express the opsins in the targeted neuronal population. In animal studies, enemas of adenoviral vectors could conceivably target enteric neurons, and surgical techniques are used to target CNS areas with the viral vectors, if not using transgenic animals with targeted opsin expression in neural sub-populations. For clinical studies, targeted viral delivery would be necessary to achieve the circuit specificity necessary for optogenetic tools, likely requiring stereotaxic targeting. The other issue with virally mediated cell transduction is the potential for immune activation. Adeno-associated viruses (AAVs) are the primary vectors that are being exploited for gene therapy studies, and would be suitable for most of the currently developed opsins (51, 83). AAVs were selected since they are not known to cause a significant immune response and different serotypes show tissue specific tropisms. However, because of their ubiquitous nature, many people have developed immune factors to AAVs, and as such concurrent short-term immunosuppression could be necessary when delivering the optogenetic vector (42, 63).

CONCLUSIONS AND FUTURE RESEARCH STRATEGIES

The future use of the techniques and tools described in this review will certainly advance our understanding of brain-gut interactions and even open new possibilities for treatment of GI disorders. The power of optogenetics to the neurogastroenterology community lies in its ability to achieve regional and cell specific neuronal modulation to affect the underlying mechanisms of disorders such as IBS. Electrophysiological approaches have proven their usefulness in measuring the electrical properties of tissues throughout the GI tract and have led to a huge growth in our understanding of the ENS, the electrical properties of the GI smooth muscle and intestinal pacemaker cells, as well as the cells within the GI epithelium. These tools will bring new treatments to patients with conditions such as IBS, functional dyspepsia, achalasia, and delayed gastric emptying.

In summary, the powerful new tools of optogenetics in combination with electrophysiological approaches will advance the field of neurogastroenterology and motility. More importantly their use will promote and encourage crosstalk and improve research collaborations between clinicians and scientists both within and outside the field of neurogastroenterology. The doors of research laboratories using these new tools and technologies should be opened to provide training of our more junior researchers to advance their careers. Furthermore, the American and European Neurogastroenterology and Motility Societies should be encouraged to provide pilot grant funding and educational opportunities to established investigators with experience in asking the right questions, to learn these contemporary approaches and bring them to their research laboratories.

GRANTS

B. Greenwood-Van Meerveld is the recipient of a Senior Research Career Scientist award (Award 1IK6BX003610-01) from the Department of Veterans Affairs. A. C. Johnson is a Career Development Awardee with the Department of Veterans Affairs (Award BX003630).

DISCLOSURES

B. Greenwood-Van Meerveld has grant funding from Ironwood Pharmaceuticals, Teva and Blue Therapeutics. This document does not represent the views of the US Department of Veteran Affairs or the US government. The remaining authors have no competing interests.

AUTHOR CONTRIBUTIONS

A.C.J. and C.O.L. prepared figures; A.C.J., T.L., C.O.L., and B.G.-V.M. drafted manuscript; A.C.J., T.L., C.O.L., and B.G.-V.M. edited and revised manuscript; A.C.J., T.L., C.O.L., and B.G.-V.M. approved final version of manuscript.