Gut enterochromaffin cells drive visceral pain and anxiety.

Gastrointestinal (GI) discomfort is a hallmark of most gut disorders and represents an important component of chronic visceral pain1. For the growing population afflicted by irritable bowel syndrome, GI hypersensitivity and pain persist long after tissue injury has resolved2. Irritable bowel syndrome also exhibits a strong sex bias, afflicting women three times more than men1. Here, we focus on enterochromaffin (EC) cells, which are rare excitable, serotonergic neuroendocrine cells in the gut epithelium3-5. EC cells detect and transduce noxious stimuli to nearby mucosal nerve endings3,6 but involvement of this signalling pathway in visceral pain and attendant sex differences has not been assessed. By enhancing or suppressing EC cell function in vivo, we show that these cells are sufficient to elicit hypersensitivity to gut distension and necessary for the sensitizing actions of isovalerate, a bacterial short-chain fatty acid associated with GI inflammation7,8. Remarkably, prolonged EC cell activation produced persistent visceral hypersensitivity, even in the absence of an instigating inflammatory episode. Furthermore, perturbing EC cell activity promoted anxiety-like behaviours which normalized after blockade of serotonergic signalling. Sex differences were noted across a range of paradigms, indicating that the EC cell-mucosal afferent circuit is tonically engaged in females. Our findings validate a critical role for EC cell-mucosal afferent signalling in acute and persistent GI pain, in addition to highlighting genetic models for studying visceral hypersensitivity and the sex bias of gut pain.

Musings on the wanderer: What's new in our understanding of vago-vagal reflexes? VI. Central vagal circuits that control glucose metabolism.

Neurons in the brain stem dorsal vagal complex (DVC) take part in a continuous bidirectional crosstalk, in which they receive and respond to a vast array of signaling molecules, including glucose. Importantly, chronic dysregulation of blood glucose concentration, a hallmark of high prevalence pathologies, such as diabetes and metabolic syndrome, can induce neuroplasticity in DVC neural networks, which is hypothesized to either contribute to or compensate for the glycemic or insulinemic dysregulation observed in these conditions. Here, we revisit the topic of vagal reflexes to review recent research on the importance of DVC function in regulating systemic glucose homeostasis and the neuroplastic changes in this brain region that are associated with systemic glucose alterations. We also discuss the critical connection between these nuclei and the gut and the role of central vagal circuits in the favorable outcomes associated with bariatric surgical procedures for metabolic disorders.

Collagen 18 and agrin are secreted by neural crest cells to remodel their microenvironment and regulate their migration during enteric nervous system development.

The enteric nervous system (ENS) arises from neural crest cells that migrate, proliferate, and differentiate into enteric neurons and glia within the intestinal wall. Many extracellular matrix (ECM) components are present in the embryonic gut, but their role in regulating ENS development is largely unknown. Here, we identify heparan sulfate proteoglycan proteins, including collagen XVIII (Col18) and agrin, as important regulators of enteric neural crest-derived cell (ENCDC) development. In developing avian hindgut, Col18 is expressed at the ENCDC wavefront, while agrin expression occurs later. Both proteins are normally present around enteric ganglia, but are absent in aganglionic gut. Using chick-mouse intestinal chimeras and enteric neurospheres, we show that vagal- and sacral-derived ENCDCs from both species secrete Col18 and agrin. Whereas glia express Col18 and agrin, enteric neurons only express the latter. Functional studies demonstrate that Col18 is permissive whereas agrin is strongly inhibitory to ENCDC migration, consistent with the timing of their expression during ENS development. We conclude that ENCDCs govern their own migration by actively remodeling their microenvironment through secretion of ECM proteins.

The digestive tract of Drosophila melanogaster.

The digestive tract plays a central role in the digestion and absorption of nutrients. Far from being a passive tube, it provides the first line of defense against pathogens and maintains energy homeostasis by exchanging neuronal and endocrine signals with other organs. Historically neglected, the gut of the fruit fly Drosophila melanogaster has recently come to the forefront of Drosophila research. Areas as diverse as stem cell biology, neurobiology, metabolism, and immunity are benefitting from the ability to study the genetics of development, growth regulation, and physiology in the same organ. In this review, we summarize our knowledge of the Drosophila digestive tract, with an emphasis on the adult midgut and its functional underpinnings.

Neural control of gut homeostasis.

The gut-brain axis is a coordinated communication system that not only maintains homeostasis, but significantly influences higher cognitive functions and emotions, as well as neurological and behavioral disorders. Among the large populations of sensory and motor neurons that innervate the gut, insights into the function of primary afferent nociceptors, whose cell bodies reside in the dorsal root ganglia and nodose ganglia, have revealed their multiple crosstalk with several cell types within the gut wall, including epithelial, vascular, and immune cells. These bidirectional communications have immunoregulatory functions, control host response to pathogens, and modulate sensations associated with gastrointestinal disorders, through activation of immune cells and glia in the peripheral and central nervous system, respectively. Here, we will review the cellular and neurochemical basis of these interactions at the periphery, in dorsal root ganglia, and in the spinal cord. We will discuss the research gaps that should be addressed to get a better understanding of the multifunctional role of sensory neurons in maintaining gut homeostasis and regulating visceral sensitivity.

Best Practice Update: Incorporating Psychogastroenterology Into Management of Digestive Disorders.

Chronic digestive diseases, including irritable bowel syndrome, gastroesophageal reflux disease, and inflammatory bowel diseases, cannot be disentangled from their psychological context-the substantial burden of these diseases is co-determined by symptom and disease severity and the ability of patients to cope with their symptoms without significant interruption to daily life. The growing field of psychogastroenterology focuses on the application of scientifically based psychological principles and techniques to the alleviation of digestive symptoms. In this Clinical Practice Update, we describe the structure and efficacy of 2 major classes of psychotherapy-cognitive behavior therapy and gut-directed hypnotherapy. We focus on the impact of these brain-gut psychotherapies on gastrointestinal symptoms, as well as their ability to facilitate improved coping, resilience, and self-regulation. The importance of the gastroenterologist in the promotion of integrated psychological care cannot be overstated, and recommendations are provided on how to address psychological issues and make an effective referral for brain-gut psychotherapy in routine practice.

Distinct or shared actions of peptide family isoforms: II. Multiple pyrokinins exert similar effects in the lobster stomatogastric nervous system.

Many neuropeptides are members of peptide families, with multiple structurally similar isoforms frequently found even within a single species. This raises the question of whether the individual peptides serve common or distinct functions. In the accompanying paper, we found high isoform specificity in the responses of the lobster (Homarus americanus) cardiac neuromuscular system to members of the pyrokinin peptide family: only one of five crustacean isoforms showed any bioactivity in the cardiac system. Because previous studies in other species had found little isoform specificity in pyrokinin actions, we examined the effects of the same five crustacean pyrokinins on the lobster stomatogastric nervous system (STNS). In contrast to our findings in the cardiac system, the effects of the five pyrokinin isoforms on the STNS were indistinguishable: they all activated or enhanced the gastric mill motor pattern, but did not alter the pyloric pattern. These results, in combination with those from the cardiac ganglion, suggest that members of a peptide family in the same species can be both isoform specific and highly promiscuous in their modulatory capacity. The mechanisms that underlie these differences in specificity have not yet been elucidated; one possible explanation, which has yet to be tested, is the presence and differential distribution of multiple receptors for members of this peptide family.

Modulation of network pacemaker neurons by oxygen at the anaerobic threshold.

Previous in vitro and in vivo studies showed that the frequency of rhythmic pyloric network activity in the lobster is modulated directly by oxygen partial pressure (PO(2)). We have extended these results by (1) increasing the period of exposure to low PO(2) and by (2) testing the sensitivity of the pyloric network to changes in PO(2) that are within the narrow range normally experienced by the lobster (1 to 6 kPa). We found that the pyloric network rhythm was indeed altered by changes in PO(2) within the range typically observed in vivo. Furthermore, a previous study showed that the lateral pyloric constrictor motor neuron (LP) contributes to the O(2) sensitivity of the pyloric network. Here, we expanded on this idea by testing the hypothesis that pyloric pacemaker neurons also contribute to pyloric O(2) sensitivity. A 2-h exposure to 1 kPa PO(2), which was twice the period used previously, decreased the frequency of an isolated group of pacemaker neurons, suggesting that changes in the rhythmogenic properties of these cells contribute to pyloric O(2) sensitivity during long-term near-anaerobic (anaerobic threshold, 0.7-1.2 kPa) conditions.

Neurodegenerative disorders and gut-brain interactions.

Neurodegenerative disorders (NDs) affect essential functions not only in the CNS, but also cause persistent gut dysfunctions, suggesting that they have an impact on both CNS and gut-innervating neurons. Although the CNS biology of NDs continues to be well studied, how gut-innervating neurons, including those that connect the gut to the brain, are affected by or involved in the etiology of these debilitating and progressive disorders has been understudied. Studies in recent years have shown how CNS and gut biology, aided by the gut-brain connecting neurons, modulate each other's functions. These studies underscore the importance of exploring the gut-innervating and gut-brain connecting neurons of the CNS and gut function in health, as well as the etiology and progression of dysfunction in NDs. In this Review, we discuss our current understanding of how the various gut-innervating neurons and gut physiology are involved in the etiology of NDs, including Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis, to cause progressive CNS and persistent gut dysfunction.

Fine mapping of the 9q31 Hirschsprung's disease locus.

Hirschsprung's disease (HSCR) is a congenital disorder characterised by the absence of ganglia along variable lengths of the intestine. The RET gene is the major HSCR gene. Reduced penetrance of RET mutations and phenotypic variability suggest the involvement of additional modifying genes in the disease. A RET-dependent modifier locus was mapped to 9q31 in families bearing no coding sequence (CDS) RET mutations. Yet, the 9q31 causative locus is to be identified. To fine-map the 9q31 region, we genotyped 301 tag-SNPs spanning 7 Mb on 137 HSCR Dutch trios. This revealed two HSCR-associated regions that were further investigated in 173 Chinese HSCR patients and 436 controls using the genotype data obtained from a genome-wide association study recently conducted. Within one of the two identified regions SVEP1 SNPs were found associated with Dutch HSCR patients in the absence of RET mutations. This ratifies the reported linkage to the 9q31 region in HSCR families with no RET CDS mutations. However, this finding could not be replicated. In Chinese, HSCR was found associated with IKBKAP. In contrast, this association was stronger in patients carrying RET CDS mutations with p = 5.10 x 10(-6) [OR = 3.32 (1.99, 5.59)] after replication. The HSCR-association found for IKBKAP in Chinese suggests population specificity and implies that RET mutation carriers may have an additional risk. Our finding is supported by the role of IKBKAP in the development of the nervous system.

Multiple mechanisms for integrating proprioceptive inputs that converge on the same motor pattern-generating network.

Movement-derived sensory feedback adapts centrally generated motor programs to changing behavioral demands. Motor circuit output may also be shaped by distinct proprioceptive systems with different central actions, although little is known about the integrative processes by which such convergent sensorimotor regulation occurs. Here, we explore the combined actions of two previously identified proprioceptors on the gastric mill motor network in the lobster stomatogastric nervous system. Both mechanoreceptors [anterior gastric receptor (AGR) and posterior stomach receptor (PSR)] access the gastric circuit via the same pair of identified projection interneurons that either excite [commissural gastric (CG)] or inhibit [gastric inhibitor (GI)] different subsets of gastric network neurons. Mechanosensory information from the two receptors is integrated upstream to the gastric circuit at two levels: (1) postsynaptically, where both receptors excite the GI neuron while exerting opposing effects on the CG neuron, and (2) presynaptically, where PSR reduces AGR's excitation of the CG projection neuron. Concomitantly PSR selectively enhances AGR's activation of the GI neuron, possibly also via a presynaptic action. PSR's influences also far outlast its transient synaptic effects, indicating the additional involvement of modulatory processes. Consequently, PSR activation causes parallel input from AGR to be conveyed preferentially via the GI interneuron, resulting in a prolonged switch in the pattern of gastric circuit output. Therefore, via a combination of short- and long-lasting, presynaptic and postsynaptic actions, one proprioceptive system is able to promote its impact on a target motor network by biasing the access of a different sensory system to the same circuit.

The receptor tyrosine kinase RET regulates hindgut colonization by sacral neural crest cells.

The enteric nervous system (ENS) is formed from vagal and sacral neural crest cells (NCC). Vagal NCC give rise to most of the ENS along the entire gut, whereas the contribution of sacral NCC is mainly limited to the hindgut. This, and data from heterotopic quail-chick grafting studies, suggests that vagal and sacral NCC have intrinsic differences in their ability to colonize the gut, and/or to respond to signalling cues within the gut environment. To better understand the molecular basis of these differences, we studied the expression of genes known to be essential for ENS formation, in sacral NCC within the chick hindgut. Our results demonstrate that, as in vagal NCC, Sox10, EdnrB, and Ret are expressed in sacral NCC within the gut. Since we did not detect a qualitative difference in expression of these ENS genes we performed DNA microarray analysis of vagal and sacral NCC. Of 11 key ENS genes examined from the total data set, Ret was the only gene identified as being highly differentially expressed, with a fourfold increase in expression in vagal versus sacral NCC. We also found that over-expression of RET in sacral NCC increased their ENS developmental potential such that larger numbers of cells entered the gut earlier in development, thus promoting the fate of sacral NCC towards that of vagal NCC.

Modulation of stomatogastric rhythms.

Neuromodulation by peptides and amines is a primary source of plasticity in the nervous system as it adapts the animal to an ever-changing environment. The crustacean stomatogastric nervous system is one of the premier systems to study neuromodulation and its effects on motor pattern generation at the cellular level. It contains the extensively modulated central pattern generators that drive the gastric mill (chewing) and pyloric (food filtering) rhythms. Neuromodulators affect all stages of neuronal processing in this system, from membrane currents and synaptic transmission in network neurons to the properties of the effector muscles. The ease with which distinct neurons are identified and their activity is recorded in this system has provided considerable insight into the mechanisms by which neuromodulators affect their target cells and modulatory neuron function. Recent evidence suggests that neuromodulators are involved in homeostatic processes and that the modulatory system itself is under modulatory control, a fascinating topic whose surface has been barely scratched. Future challenges include exploring the behavioral conditions under which these systems are activated and how their effects are regulated.

[NADPH-diaphorase activity in digestive system of gastropod molluscs Achatina fulica and Littorina littorea].

Localization and peculiarities of NO-ergic elements were studied for he first time throughout the entire length of digestive tract of the marine gastropod mollusc Achatina fulica (Prosobranchia) and the terrestrial molusc Littorina littorea (Pulmonata) by using histochemical method of detection of NADPH-diaphorase (NADPHd). NO-ergic cells and fibers were revealed in all parts of the mollusc digestive tract beginning from pharynx. An intensive NADPHd activity was found in many intraepithelial cells of the open type and in their processes in intra- and subepithelial nerve plexuses, single subepithelial neurons, granular connective tissue cells, and numerous nerve fibers among muscle elements of he digestive tract wall as well as in nerves innervating the tract. NADPHd was also present in receptor cells of he oral area and in the central A. fulica ganglia participating in innervation of the digestive tract. The digestive tract NO-ergic system ofA. fulica has a more complex organization that that of L. littorea. In the A. fulica pharynx, stomach, and midgut, directly beneath epithelium, there is revealed a complex system of glomerular structures formed by thin NADPHd-positive nerve fibers coming from the side of epithelium. More superficially under the main groups of muscle elements, small agglomerations of NADPHd-positive neurons are seen, which could be considered as primitive, non-formed microganglia. Peculiarities of distribution and a possible functional role of NO-ergic elements in the digestive tract of molluscs are discussed as compared with other invertebrate and vertebrate animals.

Oral selective serotonin reuptake inhibitors activate vagus nerve dependent gut-brain signalling.

The vagus nerve can transmit signals to the brain resulting in a reduction in depressive behavior as evidenced by the long-term beneficial effects of electrical stimulation of the vagus in patients with intractable depression. The vagus is the major neural connection between gut and brain, and we have previously shown that ingestion of beneficial bacteria modulates behaviour and brain neurochemistry via this pathway. Given the high levels of serotonin in the gut, we considered if gut-brain signaling, and specifically the vagal pathway, might contribute to the therapeutic effect of oral selective serotonin reuptake inhibitors (SSRI). Mesenteric nerve recordings were conducted in mice after treatment with SSRI to ascertain if this class of drugs resulted in increased vagal excitability. Patch clamp recordings of enteric neurons were carried out to measure activity of primary afferent neurons in the gut in response to SSRI and to assess the importance of gut epithelium in transducing signal. The tail suspension test (TST) was used following 14d feeding of SSRI in vagotomised and surgical sham mice to measure depressive-like behaviour. Brain mRNA expression was examined via PCR and the intestinal microbiome was assessed. Mesenteric nerve recordings in BALB/c mice demonstrated that oral treatment with SSRI leads to a significant increase in vagal activity. This effect was not observed in mice treated with a representative noradrenaline-dopamine reuptake inhibitor. It is known that signals from the gut can be transmitted to the vagus via the enteric nervous system. Exposure of the gut to SSRI increased the excitability of intrinsic primary afferent neurons in the myenteric plexus, through an intestinal epithelium dependent mechanism, and alpha-diversity of gut microbiota was altered. Critically, blocking vagal signaling from gut to brain, via subdiaphragmatic vagotomy, abolished the antidepressive effects of oral SSRI treatment as determined by the tail suspension test. This work suggests that vagus nerve dependent gut-brain signaling contributes to the effects of oral SSRI and further, highlights the potential for pharmacological approaches to treatment of mood disorders that focus on vagal stimulation and may not even require therapeutic agents to enter the circulation.

Orexin synthesis and response in the gut.

Orexin (hypocretin) appears to play a role in the regulation of energy balances. Previous reports have indicated that orexin-containing neurons are found only in the lateral hypothalamic (LH) area. We show that a subset of neurons in the gut which also express leptin receptors display orexin-like immunoreactivity and express functional orexin receptors. Orexin excites secretomotor neurons in the guinea pig submucosal plexus and increases motility. Moreover, fasting upregulates the phosphorylated form of cAMP response element-binding protein (pCREB) in orexin-immunoreactive neurons, indicating a functional response to food status in these cells. Together, these data suggest that orexin in the gut may play an even more intimate role in regulating energy homeostasis than it does in the CNS.

Artemin is a vascular-derived neurotropic factor for developing sympathetic neurons.

Artemin (ARTN) is a member of the GDNF family of ligands and signals through the Ret/GFRalpha3 receptor complex. Characterization of ARTN- and GFRalpha3-deficient mice revealed similar abnormalities in the migration and axonal projection pattern of the entire sympathetic nervous system. This resulted in abnormal innervation of target tissues and consequent cell death due to deficiencies of target-derived neurotrophic support. ARTN is expressed along blood vessels and in cells nearby to sympathetic axonal projections. In the developing vasculature, ARTN is expressed in smooth muscle cells of the vessels, and it acts as a guidance factor that encourages sympathetic fibers to follow blood vessels as they project toward their final target tissues. The chemoattractive properties of ARTN were confirmed by the demonstration that sympathetic neuroblasts migrate and project axons toward ARTN-soaked beads implanted into mouse embryos.

Neural crest stem cells persist in the adult gut but undergo changes in self-renewal, neuronal subtype potential, and factor responsiveness.

We found neural crest stem cells (NCSCs) in the adult gut. Postnatal gut NCSCs were isolated by flow-cytometry and compared to fetal gut NCSCs. They self-renewed extensively in culture but less than fetal gut NCSCs. Postnatal gut NCSCs made neurons that expressed a variety of neurotransmitters but lost the ability to make certain subtypes of neurons that are generated during fetal development. Postnatal gut NCSCs also differed in their responsiveness to lineage determination factors, affecting cell fate determination in vivo and possibly explaining their reduced neuronal subtype potential. These perinatal changes in gut NCSCs parallel perinatal changes in hematopoietic stem cells, suggesting that stem cells in different tissues undergo similar developmental transitions. The persistence of NCSCs in the adult PNS opens up new possibilities for regeneration after injury or disease.

Mechanosensory gating of proprioceptor input to modulatory projection neurons.

Sensorimotor gating commonly occurs at sensory neuron synapses onto motor circuit neurons and motor neurons. Here, using the crab stomatogastric nervous system, we show that sensorimotor gating also occurs at the level of the projection neurons that activate motor circuits. We compared the influence of the gastro-pyloric receptor (GPR) muscle stretch-sensitive neuron on two projection neurons, modulatory commissural neuron 1 (MCN1) and commissural projection neuron 2 (CPN2), with and without a preceding activation of the mechanosensory ventral cardiac neurons (VCNs). MCN1 and CPN2 project from the paired commissural ganglia (CoGs) to the stomatogastric ganglion (STG), where they activate the gastric mill (chewing) motor circuit. When stimulated separately, the GPR and VCN neurons each elicit the gastric mill rhythm by coactivating MCN1 and CPN2. When GPR is instead stimulated during the VCN-gastric mill rhythm, it slows this rhythm. This effect results from a second GPR synapse onto MCN1 that presynaptically inhibits its STG terminals. Here, we show that, during the VCN-triggered rhythm, the GPR excitation of MCN1 and CPN2 in the CoGs is gated out, leaving only its influence in the STG. This gating effect appears to occur within the CoG and does not result from a ceiling effect on projection neuron firing frequency. Additionally, this gating action enables GPR to either activate rhythmic motor activity or act as a phasic sensorimotor feedback system. These results also indicate that the site of sensorimotor gating can occur at the level of the projection neurons that activate a motor circuit.

Characterization of nitric oxidergic neurons in the alimentary tract of the snail Helix pomatia L.: histochemical and physiological study.

By using NADPH-diaphorase (NADPH-d) histochemistry, nitric oxide synthase (NOS) immunohistochemistry, Western blotting, and NO pharmacology, we investigated the distribution and possible function of NOS-containing neurons in different units of the alimentary tract of the snail, Helix pomatia. Discrete populations of neurons in the buccal ganglia displayed NADPH-d reactivity. NADPH-d-reactive and NOS-immunoreactive (NOS-IR) neurons were present in the caecum, and labeled fibers were found to innervate the circular muscles of the proesophagus and caecum and to form axosomatic connections with neurons of the myenteric and submucosal plexi of the caecum. A 65-kDa protein was found to be nNOS-IR in the caecum protein extract. The majority of the NADPH-d-reactive neurons also displayed FMRFamide immunoreactivity, whereas a mutual innervation by NADPH-diaphorase-reactive and catch-relaxing peptide (CARP)-IR neurons was observed in the caecum. Application of NO-donors [glyceryl trinitrate, S-nitroso-N-acetyl-DL-penicillamine, sodium nitroprusside (SNP)] evoked a dose-dependent increase in tension, frequency, and amplitude of the spontaneous muscle contractions of the proesophagus and caecum. Contractions could be blocked by applying the NO scavenger 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide. FMRFamide evoked a response of the caecum similar to that with NO, and its simultaneous application was additive. Preincubation with CARP blocked the increase of tension evoked by SNP, whereas Mytilus inhibitory peptide (MIP) decreased the rhythmic contractions induced by the NO donor. Our findings indicate that NO is an important signal molecule in the feeding system of Helix, involved, partially in cooperation with different molluscan neuropeptides, in the regulation of both neuronal and muscular activities.