Search

Background: Glucagon-like peptide-1 (GLP-1) is beneficial in relieving pain-related symptoms of Irritable bowel syndrome (IBS), a prevalent, multi-factorial functional bowel disorder characterized by diarrhea and/or constipation, abdominal bloating, and pain. Activation of myenteric neurons has been implicated in the inhibitory effects of GLP-1 on gastrointestinal motility; however, the mechanisms of action underlying this are not clear., Methods: A rat model of IBS was used to examine physiological changes evoked by intraperitoneal administration of a GLP-1 receptor agonist, exendin-4. Behavioral and physiological analysis of stress-sensitive Wister Kyoto (WKY) rats was used to determine if administration of exendin-4, in the presence or absence of neutralizing interleukin-6 receptor monoclonal antibodies, modified IBS-like symptoms. Immunofluorescence, calcium imaging, and Western blotting techniques were used to investigate the potential role of enteric neural plexi and tight junction protein expression in this effect., Key Results: Consistent with the expression of GLP-1 and interleukin-6 receptors in both submucosal and myenteric ganglia, exendin-4 and interleukin-6 stimulated calcium responses in these neurons. In vivo administration of exendin-4 normalized stress-induced defecation and visceral pain sensitivity in WKY rats. No additional changes were noted in rats co-treated with exendin-4 and anti-interleukin-6 receptor antibodies. Mucosal expression of occludin, a tight junction protein, was decreased by exendin-4. Centrally regulated anxiety-like behaviors were not modified., Conclusions and Inferences: These data suggest that intraperitoneal injection of exendin-4 improves bowel dysfunction in WKY rats without impacting on centrally regulated anxiety-like behaviors. Modulation of enteric neuronal function and tight junction expression appear to underlie the functional benefits of this intervention., (© 2019 John Wiley & Sons Ltd.)

The small intestine regulates barrier function to absorb nutrients while avoiding the entry of potentially harmful substances or bacteria. Barrier function is dynamically regulated in part by the enteric nervous system (ENS). The role of the ENS in regulating barrier function in response to luminal nutrients is not well understood. We hypothesize that the ENS regulates intestinal permeability and ion flux in the small intestine in response to luminal nutrients. Segments of jejunum and ileum from mice were mounted in Ussing chambers. Transepithelial electrical resistance (TER), short-circuit current ( I sc ), and permeability to 4-kDa FITC-dextran (FD4) were recorded after mucosal stimulation with either glucose, fructose, glutamine (10 mM), or 5% Intralipid. Mucosal lipopolysaccharide (1 mg/mL) was also studied. Enteric neurons were inhibited with tetrodotoxin (TTX; 0.5 μM) or activated with veratridine (10 μM). Enteric glia were inhibited with the connexin-43 blocker Gap26 (20 μM). Glucose, glutamine, Intralipid, and veratridine acutely modified I sc in the jejunum and ileum, but the effect of nutrients on I sc was insensitive to TTX. TTX, Gap26, and veratridine treatment did not affect baseline TER or permeability. Intralipid acutely decreased permeability to FD4, while LPS increased it. TTX pretreatment abolished the effect of Intralipid and exacerbated the LPS-induced increase in permeability. Luminal nutrients and enteric nerve activity both affect ion flux in the mouse small intestine acutely but independently of each other. Neither neuronal nor glial activity is required for the maintenance of baseline intestinal permeability; however, neuronal activity is essential for the acute regulation of intestinal permeability in response to luminal lipids and lipopolysaccharide. NEW & NOTEWORTHY Luminal nutrients and enteric nerve activity both affect ion transport in the mouse small intestine acutely, but independently of each other. Activation or inhibition of the enteric neurons does not affect intestinal permeability, but enteric neural activity is essential for the acute regulation of intestinal permeability in response to luminal lipids and lipopolysaccharide. The enteric nervous system regulates epithelial homeostasis in the small intestine in a time-dependent, region- and stimulus-specific manner.

Dopaminergic (DAergic) degeneration and abnormal α-synuclein (α-syn) expression, phosphorylation and aggregation are observed in both the nigrostriatal system (NSS) and enteric nervous system (ENS) of patients with Parkinson's disease (PD). Whether these alterations in α-syn and DAergic neurons occur synchronously in the two nervous systems or follow a process that spreads from the gut to the brain remains a subject of debate. Here, in MPTP-intoxicated cynomolgus monkeys, we showed a parallel DAergic degeneration in the colon as well as in the substantia nigra and striatum (SN/STR), as indicated by reduced expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT). In addition, we observed a simultaneous increase in the concentrations of total, phosphorylated, and oligomeric α-syn in the colon and SN/STR. Moreover, we identified that the above changes in α-syn were associated with an increase in the expression of polo-like kinase 2 (PLK2), an enzyme that promotes α-syn phosphorylation, and a decrease in the activity of protein phosphatase 2A (PP2A), an enzyme that facilitates α-syn dephosphorylation. Because the colonic ENS can be readily analyzed using routine biopsies, the shared pathological features between the colonic ENS and the brain NSS found in this study provide useful information for assessing and understanding the neuropathology in PD patients using colonic biopsies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Enteric-associated neurons (EANs) are closely associated with immune cells and continuously monitor and modulate homeostatic intestinal functions, including motility and nutrient sensing. Bidirectional interactions between neuronal and immune cells are altered during disease processes such as neurodegeneration or irritable bowel syndrome. We investigated the effects of infection-induced inflammation on intrinsic EANs (iEANs) and the role of intestinal muscularis macrophages (MMs) in this context. Using murine models of enteric infections, we observed long-term gastrointestinal symptoms, including reduced motility and loss of excitatory iEANs, which was mediated by a Nlrp6- and Casp11-dependent mechanism, depended on infection history, and could be reversed by manipulation of the microbiota. MMs responded to luminal infection by upregulating a neuroprotective program via β 2 -adrenergic receptor (β 2 -AR) signaling and mediated neuronal protection through an arginase 1-polyamine axis. Our results identify a mechanism of neuronal death post-infection and point to a role for tissue-resident MMs in limiting neuronal damage., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Mucosal barrier immunity is essential for the maintenance of the commensal microflora and combating invasive bacterial infection. Although immune and epithelial cells are thought to be the canonical orchestrators of this complex equilibrium, here, we show that the enteric nervous system (ENS) plays an essential and non-redundant role in governing the antimicrobial protein (AMP) response. Using confocal microscopy and single-molecule fluorescence in situ mRNA hybridization (smFISH) studies, we observed that intestinal neurons produce the pleiotropic cytokine IL-18. Strikingly, deletion of IL-18 from the enteric neurons alone, but not immune or epithelial cells, rendered mice susceptible to invasive Salmonella typhimurium (S.t.) infection. Mechanistically, unbiased RNA sequencing and single-cell sequencing revealed that enteric neuronal IL-18 is specifically required for homeostatic goblet cell AMP production. Together, we show that neuron-derived IL-18 signaling controls tissue-wide intestinal immunity and has profound consequences on the mucosal barrier and invasive bacterial killing., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Ulcerative colitis (UC) and Crohn's disease (CD) are pathological conditions with an unknown aetiology that are characterised by severe inflammation of the intestinal tract and collectively referred to as inflammatory bowel disease (IBD). Current treatments are mostly ineffective due to their limited efficacy or toxicity, necessitating surgical resection of the affected bowel. The management of IBD is hindered by a lack of prognostic markers for clinical inflammatory relapse. Intestinal inflammation associates with the infiltration of immune cells (leukocytes) into, or surrounding the neuronal ganglia of the enteric nervous system (ENS) termed plexitis or ganglionitis. Histological observation of plexitis in unaffected intestinal regions is emerging as a vital predictive marker for IBD relapses. Plexitis associates with alterations to the structure, cellular composition, molecular expression and electrophysiological function of enteric neurons. Moreover, plexitis often occurs before the onset of gross clinical inflammation, which may indicate that plexitis can contribute to the progression of intestinal inflammation. In this review, the bilateral relationships between the ENS and inflammation are discussed. These include the effects and mechanisms of inflammation-induced enteric neuronal loss and plasticity. Additionally, the role of enteric neurons in preventing antigenic/pathogenic insult and immunomodulation is explored. While all current treatments target the inflammatory pathology of IBD, interventions that protect the ENS may offer an alternative avenue for therapeutic intervention., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The gut-brain axis is a hot topic in Parkinson's disease. In an attempt to decipher its role in the disease pathogenesis, several animal models have been developed. Most of these models tried to reproduce Braak's hypothesis by showing that the pathological process could spread from the gut to the brain (bottom-up scenario). Interestingly, others groups showed that a top-down scenario could also occur and that 6-hydroxydopamine-induced nigrostriatal denervation was sufficient to induce significant changes in the gastrointestinal tract. Aside from this toxic approach, the article by O'Donovan and colleagues in this edition of Neurogastroenterology and Motility showed that bilateral nigral injection of adeno-associated virus (AAV)-alpha-synuclein in rats was accompanied by changes in the enteric nervous system and the gut microbiota, which occurred without any apparent brain-to-gut spread of human injected alpha-synuclein. Some changes observed in the gastrointestinal tract of animals injected with AAV-alpha-synuclein were in line with previous observations in Parkinson's disease patients, including increased expression of glial markers, swollen tyrosine hydroxylase-positive varicosities in the submucosal plexus, and decreases in Faecalibacterium and Lachnospiraceae. These findings suggest that, in addition to gut-brain pathways, the brain-to-gut communication may also be involved in Parkinson's disease pathophysiology. In this mini-review, we describe the strengths and limitations of the existing studies on the gut-brain axis in experimental models of parkinsonism and discuss an alternative hypothesis in which the central and enteric nervous system would evolve separately during disease progression., (© 2019 John Wiley & Sons Ltd.)

Background & Aims: Functional interactions between the mu opioid receptor (MOR) and delta opioid receptor (DOR) represent a potential target for novel analgesics and may drive the effects of the clinically approved drug eluxadoline for the treatment of diarrhea-predominant irritable bowel syndrome. Although the enteric nervous system (ENS) is a likely site of action, the coexpression and potential interaction between MOR and DOR in the ENS are largely undefined. In the present study, we have characterized the distribution of MOR in the mouse ENS and examined MOR-DOR interactions by using pharmacologic and cell biology techniques., Methods: MOR and DOR expression was defined by using MORmCherry and MORmCherry-DOR-eGFP knockin mice. MOR-DOR interactions were assessed by using DOR-eGFP internalization assays and by pharmacologic analysis of neurogenic contractions of the colon., Results: Although MOR was expressed by approximately half of all myenteric neurons, MOR-positive submucosal neurons were rarely observed. There was extensive overlap between MOR and DOR in both excitatory and inhibitory pathways involved in the coordination of intestinal motility. MOR and DOR can functionally interact, as shown through heterologous desensitization of MOR-dependent responses by DOR agonists. Functional evidence suggests that MOR and DOR may not exist as heteromers in the ENS. Pharmacologic studies show no evidence of cooperativity between MOR and DOR. DOR internalizes independently of MOR in myenteric neurons, and MOR-evoked contractions are unaffected by the sequestration of DOR., Conclusions: Collectively, these findings demonstrate that although MOR and DOR are coexpressed in the ENS and functionally interact, they are unlikely to exist as heteromers under physiological conditions., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Gastrointestinal (GI) comorbidities are common in individuals with mood and behavioral dysfunction. Similarly, patients with GI problems more commonly suffer from co-morbid psychiatric diagnoses. Although the central and enteric nervous systems (CNS and ENS, respectively) have largely been studied separately, there is emerging interest in factors that may contribute to disease states involving both systems. There is strong evidence to suggest that serotonin may be an important contributor to these brain-gut conditions. Serotonin has long been recognized for its critical functions in CNS development and function. The majority of the body's serotonin, however, is produced in the GI tract, where it plays key roles in ENS development and function. Further understanding of the specific impact that enteric serotonin has on brain-gut disease may lay the foundation for the creation of novel therapeutic targets. This review summarizes the current data focusing on the important roles that serotonin plays in ENS development and motility, with a focus on novel aspects of serotonergic signaling in medical conditions in which CNS and ENS co-morbidities are common, including autism spectrum disorders and depression.

Zinc oxide nanoparticles (ZnONPs) have been widely used in food storage containers and food additives in daily life. However, the impact of oral intake of ZnONPs on nervous system is extremely limited, especially on children and adolescents. In this study, four weeks old mice were treated with either vehicle or ZnONPs suspension solution at 26 mg/kg by intragastric administration for 30 days. Our results demonstrated that oral ZnONPs exposure could induce pathological changes in gut and abnormal excitement of enteric neurons. Interestingly, we found that ZnONPs caused enhancement of 5-hydroxytryptamine (5-HT) in gut by activation of its biosynthesis, transport and receptors, and subsequently resulting in increased level of 5-HT in brain via gut-brain communication by blood. Our data also showed that there were no apparent changes on the expressions of interleukin (Il)-6, Il-1β, C-C motif chemokine ligand 2 (Ccl2), tumor necrosis factor (Tnf) in gut and zinc chelator Mt2 in gut and cortex. Meanwhile, no significant changes were observed on the expressions of tryptophan hydroxylase type 1, 5-HT receptor 3A (Htr3a) and Htr4 in hippocampus and cortex. Our study indicate that oral ZnONPs exposure causes hyperfunction of 5-HT in gut in young mice which may further spread to brain via gut-brain communication., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Alpha-Synuclein (α-Syn) is expressed in the central nervous system and the nervous system of the gut (enteric nervous system, ENS), and is well known to be the major constituent of Lewy bodies which are the hallmark of Parkinson's disease. Gastrointestinal disorders frequently manifest several years before motor deficits develop in Parkinson's patients. Despite extensive research on pathological rodent models, the physiological role of α-Syn in the normal ENS is unclear hampering analysis of its neuropathology. We compared the ENS in colons of α-Syn-knockout (α-Syn KO) and wild-type mice using immunohistochemistry and calcium-imaging of responses to synaptic input. We found that α-Syn is predominantly expressed in cholinergic varicosities, which contain vesicular acetylcholine transporter. α-Syn KO mice had higher enteric neuron density and a larger proportion of cholinergic neurons, notably those containing calretinin, demonstrating a role for α-Syn in regulating development of these neurons. Moreover, α-Syn deletion enhanced the amplitude of synaptically activated [Ca 2+ ] i transients that are primarily mediated by acetylcholine activating nicotinic receptors suggesting that α-Syn modulates the availability of acetylcholine in enteric nerve terminals., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Sialylated milk oligosaccharides (SMOs) have a multifunctional health benefit, yet the molecular details underlying their potential role in modulating intestinal maturation remains unknown. To test the hypothesis that sialyllactose (SL) may mediate intestinal maturation and function through controlling neuronal function, studies were carried out where the diet of postnatal piglets was supplemented with a mixture of 3'- and 6'-sialyllactose from postnatal day 3 to 38. Gene transcription pathways regulating enteric nervous system function, polysialic acid (polySia) synthesis, and cell proliferation were quantified. Our new findings show that SL intervention: (1) upregulated the level of gene and protein expression of the glial-derived neurotrophic factor (GDNF) in the ileum; (2) upregulated phosphorylation of the cAMP responsive element-binding protein (CREB), the downstream target of GDNF signaling pathway; (3) promoted cell proliferation based on an increase in the number and density of Ki-67 positive cells in the crypts; (4) increased the crypt width in the ileum by 10%, while gene markers for the functional cells were not affected; (5) upregulated mRNA expression level of ST8Sia IV, a key polysialyltransferase responsible for synthesis of polySia-NCAM; (6) reduced the incidence and severity of diarrhea. These results show that SL promotes intestinal maturation in neonatal piglets by upregulating GDNF, synthesis of polySia and CREB-interactive pathway.

Background: Cystic fibrosis transmembrane conductance regulator (CFTR) was recently found in the enteric nervous system, where its role is unclear. We aimed to identify which enteric neuronal structures express CFTR, whether CFTR modulates enteric neurotransmission and if altered CFTR expression is associated with slow transit constipation (STC)., Methods: Immunofluorescence double labeling was performed to localize CFTR with various neuronal and glial cell markers in the human colon. The immunoreactivity (IR) of CFTR and choline acetyltransferase (ChAT) on myenteric plexus of control and STC colon was quantitatively analyzed. In control colonic muscle strips, electrical field stimulation (EFS) evoked contractile responses and the release of acetylcholine (ACh) was measured in the presence of the CFTR channel inhibitor, CFTR(inh)-172., Results: CFTR-IR was densely localized to myenteric ganglia, where it was co-localized with neuronal markers HuC/D and β-tubulin, and glial marker S-100 but little with glial fibrillary acidic protein. Vesicular ACh transport was almost exclusively co-localized with CFTR, but neurons expressing nitric oxide synthase were CFTR negative. Significant reductions of CFTR-IR (P < 0.01) and ChAT-IR (P < 0.05) were observed on myenteric ganglia of STC compared to control. Pre-treatment of colonic muscle strips with CFTR(inh)-172 (10 µM) significantly inhibited EFS-evoked contractile responses (P < 0.01) and ACh release (P < 0.05)., Conclusions: Co-localization of CFTR-IR with cholinergic markers, inhibition of EFS-induced colonic muscle contractility and ACh release by CFTR(inh)-172 suggest that CFTR modulates enteric cholinergic neurotransmission. The downregulation of CFTR and ChAT in myenteric ganglia of STC correlated with the impaired contractile responses to EFS.

The importance of the gut-brain axis in maintaining homeostasis has long been appreciated. However, the past 15 yr have seen the emergence of the microbiota (the trillions of microorganisms within and on our bodies) as one of the key regulators of gut-brain function and has led to the appreciation of the importance of a distinct microbiota-gut-brain axis. This axis is gaining ever more traction in fields investigating the biological and physiological basis of psychiatric, neurodevelopmental, age-related, and neurodegenerative disorders. The microbiota and the brain communicate with each other via various routes including the immune system, tryptophan metabolism, the vagus nerve and the enteric nervous system, involving microbial metabolites such as short-chain fatty acids, branched chain amino acids, and peptidoglycans. Many factors can influence microbiota composition in early life, including infection, mode of birth delivery, use of antibiotic medications, the nature of nutritional provision, environmental stressors, and host genetics. At the other extreme of life, microbial diversity diminishes with aging. Stress, in particular, can significantly impact the microbiota-gut-brain axis at all stages of life. Much recent work has implicated the gut microbiota in many conditions including autism, anxiety, obesity, schizophrenia, Parkinson's disease, and Alzheimer's disease. Animal models have been paramount in linking the regulation of fundamental neural processes, such as neurogenesis and myelination, to microbiome activation of microglia. Moreover, translational human studies are ongoing and will greatly enhance the field. Future studies will focus on understanding the mechanisms underlying the microbiota-gut-brain axis and attempt to elucidate microbial-based intervention and therapeutic strategies for neuropsychiatric disorders.

Background: The enteric nervous system (ENS), a complex network of neurons and glial cells, coordinates major gastrointestinal functions. Impaired development or secondary aberrations cause severe enteric neuropathies. Neural crest-derived stem cells as well as enteric neuronal progenitor cells, which form enteric neurospheres, represent a promising tool to unravel molecular pathomechanisms and to develop novel therapy options. However, so far little is known about the detailed cellular composition and the proportional distribution of enteric neurospheres. Comprehensive knowledge will not only be essential for basic research but also for prospective cell replacement therapies to restore or to improve enteric neuronal dysfunction., Methods: Human enteric neurospheres were generated from three individuals with varying age. For detailed molecular characterization, nCounter target gene expression analyses focusing on stem, progenitor, neuronal, glial, muscular, and epithelial cell markers were performed. Corresponding archived paraffin-embedded individuals' specimens were analyzed accordingly., Key Results: Our data revealed a remarkable molecular complexity of enteric neurospheres and archived specimens. Amongst the expression of multipotent stem cell, progenitor cell, neuronal, glial, muscle and epithelial cell markers, moderate levels for the pluripotency marker POU5F1 were observed. Furthermore, besides the interindividual variability, we identified highly distinct intraindividual expression profiles., Conclusions & Inferences: Our results emphasize the assessment of molecular signatures to be essential for standardized use, optimization of experimental approaches, and elimination of potential risk factors, as the formation of tumors. Our study pipeline may serve as a blueprint implemented into the characterization procedure of enteric neurospheres for various future applications., (© 2019 John Wiley & Sons Ltd.)

Background: The enteric nervous system contains inhibitory and excitatory motor neurons which modulate smooth muscle contractility. Cell bodies of longitudinal muscle motor neurons have not been identified in human intestine., Methods: We used retrograde tracing ex vivo with DiI, with multiple labeling immunohistochemistry, to characterize motor neurons innervating tenial and inter-tenial longitudinal muscle of human colon., Key Results: The most abundant immunohistochemical markers in the tertiary plexus were vesicular acetylcholine transporter, nitric oxide synthase (NOS), and vasoactive intestinal polypeptide (VIP). Of retrogradely traced motor neurons innervating inter-tenial longitudinal muscle, 95% were located within 6mm oral or anal to the DiI application site. Excitatory motor neuron cell bodies, immunoreactive for choline acetyltransferase (ChAT), were clustered aborally, whereas NOS-immunoreactive cell bodies were distributed either side of the DiI application site. Motor neurons had small cell bodies, averaging 438 + 18µm 2 in cross-sectional area, similar for ChAT- and NOS-immunoreactive subtypes. Motor neurons innervating the tenia had slightly longer axial projections, with 95% located within 9mm. ChAT-immunoreactive excitatory motor neurons to tenia were clustered aborally, whereas NOS-immunoreactive inhibitory motor neurons had both ascending and descending projections. VIP immunoreactivity was rarely present without NOS immunoreactivity in motor neurons., Conclusions and Inferences: Tenial and inter-tenial motor neurons innervating the longitudinal muscle have short projections. Inhibitory motor neurons have less polarized projections than cholinergic excitatory motor neurons. Longitudinal and circular muscle layers are innervated by distinct local populations of excitatory and inhibitory motor neurons. A population of human enteric neurons that contribute significantly to colonic motility has been characterized., (© 2019 John Wiley & Sons Ltd.)

Somatostatin (SOM) is an active substance which most commonly occurs in endocrine cells, as well as in the central and peripheral nervous system. One of the parts of the nervous system where the presence of SOM has been confirmed is the enteric nervous system (ENS), located in the wall of the gastrointestinal (GI) tract. It regulates most of the functions of the stomach and intestine and it is characterized by complex organization and a high degree of independence from the central nervous system. SOM has been described in the ENS of numerous mammal species and its main functions in the GI tract are connected with the inhibition of the intestinal motility and secretory activity. Moreover, SOM participates in sensory and pain stimuli conduction, modulation of the release of other neuronal factors, and regulation of blood flow in the intestinal vessels. This peptide is also involved in the pathological processes in the GI tract and is known as an anti-inflammatory agent. This paper, which focuses primarily on the distribution of SOM in the ENS and extrinsic intestinal innervation in various mammalian species, is a review of studies concerning this issue published from 1973 to the present.

Parkinson's disease (PD) is a complex neurological disorder classically characterized by impairments in motor system function associated with loss of dopaminergic neurons in the substantia nigra. After almost 200 years since the first description of PD by James Parkinson, unraveling the complexity of PD continues to evolve. It is now recognized that an interplay between genetic and environmental factors influences a diverse range of cellular processes, reflecting on other clinical features including non-motor symptoms. This has consequently highlighted the extensive value of early clinical diagnosis to reduce difficulties of later stage management of PD. Advancement in understanding of PD has made remarkable progress in introducing new tools and strategies such as stem cell therapy and deep brain stimulation. A link between alterations in gut microbiota and PD has also opened a new line. Evidence exists of a bidirectional pathway between the gastrointestinal tract and the central nervous system. Probiotics, prebiotics and synbiotics are being examined that might influence gut-brain axis by altering gut microbiota composition, enteric nervous system, and CNS. This review provides status on use of probiotics for PD. Limitations and future directions will also be addressed to promote further research considering use of probiotics for PD., Competing Interests: The “Probiotic Research Initiative™” is a collaboration between Bifodan A/S (Hundested, Denmark) and the Biomedicine group at Aalborg University. The author is affiliated with the Biomedicine group at Aalborg University and, in collaboration with Bifodan A/S, has performed research related to applying the probiotic Lactobacillus rhamnosus PB01 (DSM 14870) produced by Bifodan A/S in several domains including pain, obesity, and fertility [162,163].

Since several years, the enteric nervous system (ENS) is getting more and more in the focus of gastrointestinal research. While the main interest was credited for years to the enteric neurons and their functional properties, less attention has been paid on the enteric glial cells (EGCs). Although the similarity of EGCs to central nervous system (CNS) astrocytes has been demonstrated a long time ago, EGCs were investigated in more detail only recently. Similar to the CNS, there is not "the" EGC, but also a broad range of diversity. Based on morphology and protein expression, such as glial fibrillary acidic protein (GFAP), S100, or Proteolipid-protein-1 (PLP1), several distinct glial types can be differentiated. Their heterogeneity in morphology, localization, and transcription as well as interaction with surrounding cells indicate versatile functional properties of these cells for gut function in health and disease. Although NG2 is found in a subset of CNS glial cells, it did not colocalize with the glial marker S100 or GFAP in the ENS. Instead, it in part colocalize with PDGFRα, as it does in the CNS, which do stain fibroblast-like cells in the gastrointestinal tract. Moreover, there seem to be species dependent differences. While GFAP is always found in the rodent ENS, this is completely different for the human gut. Only the compromised human ENS shows a significant amount of GFAP-positive glial cells. So, in general we can conclude that the EGC population is species specific and as complex as CNS glia. Anat Rec, 302:1333-1344, 2019. © 2019 Wiley Periodicals, Inc., (© 2019 Wiley Periodicals, Inc.)

Endocytosis is a major mechanism through which cellular signaling by G protein-coupled receptors (GPCRs) is terminated. However, recent studies demonstrate that GPCRs are internalized in an active state and continue to signal from within endosomes, resulting in effects on cellular function that are distinct to those arising at the cell surface. Endocytosis inhibitors are commonly used to define the importance of GPCR internalization for physiological and pathophysiological processes. Here, we provide the first detailed examination of the effects of these inhibitors on neurogenic contractions of gastrointestinal smooth muscle, a key preliminary step to evaluate the importance of GPCR endocytosis for gut function. Inhibitors of clathrin-mediated endocytosis (Pitstop2, PS2) or G protein-coupled receptor kinase-2/3-dependent phosphorylation (Takeda compound 101, Cmpd101), significantly reduced GPCR internalization. However, they also attenuated cholinergic contractions through different mechanisms. PS2 abolished contractile responses by colonic muscle to SNC80 and morphine, which strongly and weakly internalize δ-opioid and μ-opioid receptors, respectively. PS2 did not affect the increased myogenic contractile activity following removal of an inhibitory neural influence (tetrodotoxin) but suppressed electrically evoked neurogenic contractions. Ca 2+ signaling by myenteric neurons in response to exogenous ATP was unaffected by PS2, suggesting inhibitory actions on neurotransmitter release rather than neurotransmission. In contrast, Cmpd101 attenuated contractions to the cholinergic agonist carbachol, indicating direct effects on smooth muscle. We conclude that, although PS2 and Cmpd101 are effective blockers of GPCR endocytosis in enteric neurons, these inhibitors are unsuitable for the study of neurally mediated gut function due to their inhibitory effects on neuromuscular transmission and smooth muscle contractility. NEW & NOTEWORTHY Internalization of activated G protein-coupled receptors is a major determinant of the type and duration of subsequent downstream signaling events. Inhibitors of endocytosis effectively block opioid receptor internalization in enteric neurons. The clathrin-dependent endocytosis inhibitor Pitstop2 blocks effects of opioids on neurogenic contractions of the colon in an internalization-independent manner. These inhibitors also significantly impact cholinergic neuromuscular transmission. We conclude that these tools are unsuitable for examination of the contribution of neuronal G protein-coupled receptor endocytosis to gastrointestinal motility.

Background: Gastrointestinal (GI) symptoms are common in Parkinson disease (PD), often preceding neurological manifestations; however, early diagnostic utility of GI biopsies remains controversial. Studies suggest aberrant deposition of alpha-synuclein (α-syn) follows step-wise progression in central nervous system though histologic interpretation of normal and aberrant staining patterns have shown variable results. This study examines whether GI α-syn mRNA expression combined with standard α-syn immunohistochemical staining enhance the role of GI biopsy in PD., Materials and Methods: Four groups were examined, including pediatric (21) and adult control patients (18), PD clinic patients (17), and pathologically confirmed PD cases from hospital archives (16). Enteric nervous system α-syn staining was evaluated by immunohistochemistry in 33 PD and 39 controls. α-Syn mRNA levels were compared between patient groups using quantitative polymerase chain reaction and stomach and colon levels in PD., Results: PD patients had Lewy bodies (LB) and diffuse neuronal α-syn staining. GI tissues from elderly controls, children, and young adults exhibited diffuse positivity. LB were limited to PD. Myenteric plexus immunoreactivity varied in different regions. Widespread staining was noted within stomach and colon. Immunoreactivity was present within esophagus, appendix, and small bowel. α-Syn mRNA expression was highest in PD; however, levels varied between proximal and distal GI tract., Conclusions: α-Syn is normally present within young and elderly enteric nervous system; furthermore, while α-syn mRNA is always detectable, levels are highest and most variable in PD. This suggests that enteric α-syn may be altered in neurodegenerative disease. The presence of LB in the GI tract, not solely α-syn expression, may prove useful, distinguishing neurodegenerative disease patients from normal controls.

Identification and isolation of neural progenitor cells from the human enteric nervous system (ENS) is currently hampered by the lack of reliable, specific markers. Here, we define the Wnt-receptor frizzled-4 as a marker for the isolation of enteric neural progenitor cells derived from paediatric gut samples. We show that the Wnt-receptor frizzled-4 is expressed in the human colon and in Tunica muscularis -derived enterospheres. To obtain a purified culture, we carried out fluorescence-activated cell sorting (FACS) using PE-conjugated frizzled-4 antibodies. Frizzled-4 positive cells gave rise to neurosphere-like bodies and ultimately differentiated into neurons as revealed by BrdU-proliferation assays and immunocytochemistry, whereas in frizzled-4 negative cultures we did not detect any neuronal and glial cells. By using a patch-clamp approach, we also demonstrated the expression of functional sodium and potassium channels in frizzled-4 positive cell cultures after differentiation in vitro.

The identification and characterization of fungal commensals of the human gut (the mycobiota) is ongoing, and the effects of their various secondary metabolites on the health and disease of the host is a matter of current research. While the neurons of the central nervous system might be affected indirectly by compounds from gut microorganisms, the largest peripheral neuronal network (the enteric nervous system) is located within the gut and is exposed directly to such metabolites. We analyzed 320 fungal extracts and their effect on the viability of a human neuronal cell line (SH-SY5Y), as well as their effects on the viability and functionality of the most effective compound on primary enteric neurons of murine origin. An extract from P. coprobium was identified to decrease viability with an EC 50 of 0.23 ng/µL in SH-SY5Y cells and an EC 50 of 1 ng/µL in enteric neurons. Further spectral analysis revealed that the effective compound was patulin, and that this polyketide lactone is not only capable of evoking ROS production in SH-SY5Y cells, but also diverse functional disabilities in primary enteric neurons such as altered calcium signaling. As patulin can be found as a common contaminant on fruit and vegetables and causes intestinal injury, deciphering its specific impact on enteric neurons might help in the elaboration of preventive strategies., Competing Interests: The authors declare no conflict of interest.

Background & Aims: Reduced gastrointestinal (GI) motility is a feature of disorders associated with intestinal dysbiosis and loss of beneficial microbes. It is not clear how consumption of beneficial commensal microbes, marketed as probiotics, affects the enteric nervous system (ENS). We studied the effects of the widely used probiotic and the commensal Lactobacillus rhamnosus GG (LGG) on ENS and GI motility in mice., Methods: Conventional and germ free C57B6 mice were gavaged with LGG and intestinal tissues were collected; changes in the enteric neuronal subtypes were assessed by real-time polymerase chain reaction, immunoblots, and immunostaining. Production of reactive oxygen species (ROS) in the jejunal myenteric plexi and phosphorylation (p) of mitogen-activated protein kinase 1 (MAPK1) in the enteric ganglia were assessed by immunoblots and immunostaining. Fluorescence in situ hybridization was performed on jejunal cryosections with probes to detect formyl peptide receptor 1 (FPR1). GI motility in conventional mice was assessed after daily gavage of LGG for 1 week., Results: Feeding of LGG to mice stimulated myenteric production of ROS, increased levels of phosphorylated MAPK1, and increased expression of choline acetyl transferase by neurons (P < .001). These effects were not observed in mice given N-acetyl cysteine (a ROS inhibitor) or LGGΩSpaC (an adhesion-mutant strain of LGG) or FPR1-knockout mice. Gavage of mice with LGG for 1 week significantly increased stool frequency, reduced total GI transit time, and increased contractions of ileal circular muscle strips in ex vivo experiments (P < .05)., Conclusions: Using mouse models, we found that LGG-mediated signaling in the ENS requires bacterial adhesion, redox mechanisms, and FPR1. This pathway might be activated to increase GI motility in patients., (Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Xenin-25 is a neurotensin-like peptide that is secreted by enteroendocrine cells in the small intestine. Xenin-8 is reported to augment duodenal anion secretion by activating afferent neural pathways. The intrinsic neuronal circuits mediating the xenin-25-induced anion secretion were characterized using the Ussing-chambered, mucosa-submucosa preparation from the rat ileum. Serosal application of xenin-25 increased the short-circuit current in a concentration-dependent manner. The responses were abolished by the combination of Cl - -free and HCO 3 - -free solutions. The responses were almost completely blocked by TTX (10 -6 M) but not by atropine (10 -5 M) or hexamethonium (10 -4 M). The selective antagonists for neurotensin receptor 1 (NTSR1), neurokinin 1 (NK1), vasoactive intestinal polypeptide (VIP) receptors 1 and 2 (VPAC1 and VPAC2, respectively), and capsaicin, but not 5-hydroxyltryptamine receptors 3 and 4 (5-HT 3 and 5-HT 4 ), NTSR2, and A803467, inhibited the responses to xenin-25. The expression of VIP receptors ( Vipr ) in rat ileum was examined using RT-PCR. The Vipr1 PCR products were detected in the submucosal plexus and mucosa. Immunohistochemical staining showed the colocalization of NTSR1 and NK1 with substance P (SP)- and calbindin-immunoreactive neurons in the submucosal plexus, respectively. In addition, NK1 was colocalized with noncholinergic VIP secretomotor neurons. Based on the results from the present study, xenin-25-induced Cl - / HCO 3 - secretion is involved in NTSR1 activation on intrinsic and extrinsic afferent neurons, followed by the release of SP and subsequent activation of NK1 expressed on noncholinergic VIP secretomotor neurons. Finally, the secreted VIP may activate VPAC1 on epithelial cells to induce Cl - / HCO 3 - secretion in the rat ileum. Activation of noncholinergic VIP secretomotor neurons by intrinsic primary afferent neurons and extrinsic afferent neurons by postprandially released xenin-25 may account for most of the neurogenic secretory response induced by xenin-25. NEW & NOTEWORTHY This study is the first to investigate the intrinsic neuronal circuit responsible for xenin-25-induced anion secretion in the rat small intestine. We have found that nutrient-stimulated xenin-25 release may activate noncholinergic vasoactive intestinal polypeptide (VIP) secretomotor neurons to promote Cl - / HCO 3 - secretion through the activation of VIP receptor 1 on epithelial cells. Moreover, the xenin-25-induced secretory responses are mainly linked with intrinsic primary afferent neurons, which are involved in the activation of neurotensin receptor 1 and neurokinin 1 receptor.

Background: Bisphenol A (BPA) is commonly used in the production of plastics and has multidirectional, negative effects on the living organisms. It may also affect the enteric nervous system (ENS) located in the wall of the gastrointestinal tract. Enteric neurons express many active substances, which regulate majority of intestinal activities not only in physiological conditions but also under the impact of pathological factors., Methods: The influence of various doses of BPA on the ENS of jejunum has been investigated using the double immunofluorescence technique. The commercial antibodies against substance P (SP), vasoactive intestinal polypeptide (VIP), galanin (GAL), vesicular acetylcholine transporter (VAChT), and cocaine- and amphetamine-regulated transcript peptide (CART) were used., Key Results: Both doses of BPA studied changed the number of the enteric neurons immunoreactive to SP, VIP, GAL, VAChT, and CART, and the intensity of fluctuations depended on the BPA dose and on the type of the enteric plexus. Bisphenol A causes the increase in the number of neurons immunoreactive to the majority of substances studied. The only exception was VAChT-positive neurons, the number of which was lower under the impact of BPA in the comparison with physiological conditions., Conclusions & Inferences: Even low doses of BPA cause the changes in neurochemical characterization of the enteric neurons in the jejunum. These changes may be the first sign of subclinical BPA intoxication. The mechanisms of observed changes are probably connected with neurotoxic and/or pro-inflammatory activity of BPA, but their exact mechanisms are not fully explained., (© 2019 John Wiley & Sons Ltd.)

Parkinson's disease (PD), the second most common neurodegenerative movement disorder, is characterized by progressive motor and non-motor symptoms [1]. Despite treatment with pharmacologic and surgical therapies, the disease will continue to relentlessly advance. Hence, there is a great deal of interest in potential disease-modifying therapies with the hope that the neurodegenerative process can be slowed or halted. The purpose of this review is to highlight the role toxic α-synuclein (α-syn) plays in PD pathogenesis and critically review the relevant literature about therapeutic modalities targeting α-syn. Toxic α-syn plays a key role in PD pathogenesis, disrupting important cellular functions, and, thus, targeting α-syn is a reasonable disease-modifying strategy. Current approaches under investigation include decreasing α-syn production with RNA interference (RNAi), inhibiting α-syn aggregation, promoting intracellular degradation of α-syn aggregates (via enhancing autophagy and enhancing lysosomal degradation), and promoting extracellular degradation of α-syn via active and passive immunization.

Altered intestinal permeability has been correlated with Parkinson's pathophysiology in the enteric nervous system, before manifestations in the central nervous system (CNS). The inflammatory endotoxin or lipopolysaccharide (LPS) released by gut bacteria is known to modulate α-synuclein amyloidogenesis through the formation of intermediate nucleating species. Here, biophysical techniques in conjunction with microscopic images revealed the molecular interaction between lipopolysaccharide and α-synuclein that induce rapid nucleation events. This heteromolecular interaction stabilizes the α-helical intermediates in the α-synuclein aggregation pathway. Multitude NMR studies probed the residues involved in the LPS-binding structural motif that modulates the nucleating forms, affecting the cellular internalization and associated cytotoxicity. Collectively, our data characterizes this heteromolecular interaction associated with an alternative pathway in Parkinson's disease progression.

The gastrointestinal tract is constructed with an intrinsic series of interconnected ganglia that span its entire length, called the enteric nervous system (ENS). The ENS exerts critical local reflex control over many essential gut functions; including peristalsis, water balance, hormone secretions and intestinal barrier homeostasis. ENS ganglia exist as a collection of neurons and glia that are arranged in a series of plexuses throughout the gut: the myenteric plexus and submucosal plexus. While it is known that enteric ganglia are derived from a stem cell population called the neural crest, mechanisms that dictate final neuropil plexus organization remain obscure. Recently, the vertebrate animal, zebrafish, has emerged as a useful model to understand ENS development, however knowledge of its developing myenteric plexus architecture was unknown. Here, we examine myenteric plexus of the maturing zebrafish larval fish histologically over time and find that it consists of a series of tight axon layers and long glial cell processes that wrap the circumference of the gut tube to completely encapsulate it, along all levels of the gut. By late larval stages, complexity of the myenteric plexus increases such that a layer of axons is juxtaposed to concentric layers of glial cells. Ultrastructurally, glial cells contain glial filaments and make intimate contacts with one another in long, thread-like projections. Conserved indicators of vesicular axon profiles are readily abundant throughout the larval plexus neuropil. Together, these data extend our understanding of myenteric plexus architecture in maturing zebrafish, thereby enabling functional studies of its formation in the future.

Medullary thyroid carcinoma (MTC) develops from hyperplasia of thyroid C cells and represents one of the major causes of thyroid cancer mortality. Mutations in the cysteine-rich domain (CRD) of the RET gene are the most prevalent genetic cause of MTC. The current consensus holds that such cysteine mutations cause ligand-independent dimerization and constitutive activation of RET. However, given the number of the CRD mutations left uncharacterized, our understanding of the pathogenetic mechanisms by which CRD mutations lead to MTC remains incomplete. We report here that RET(C618F), a mutation identified in MTC patients, displays moderately high basal activity and requires the ligand for its full activation. To assess the biological significance of RET(C618F) in organogenesis, we generated a knock-in mouse line conditionally expressing RET(C618F) cDNA by the Ret promoter. The RET(C618F) allele can be made to be Ret-null and express mCherry by Cre-loxP recombination, which allows the assessment of the biological influence of RET(C618F) in vivo. Mice expressing RET(C618F) display mild C cell hyperplasia and increased numbers of enteric neurons, indicating that RET(C618F) confers gain-of-function phenotypes. This mouse line serves as a novel biological platform for investigating pathogenetic mechanisms involved in MTC and enteric hyperganglionosis., (© 2019 Wiley Periodicals, Inc.)

Normal gut function relies on the activity of the enteric nervous system (ENS) found within the wall of the gastrointestinal tract. The structural and functional organization of the ENS has been extensively studied in the guinea pig small intestine, but less is known about colonic circuitry. Given that there are significant differences between these regions in function, observed motor patterns and pathology, it would be valuable to have a better understanding of the colonic ENS. Furthermore, disorders of colonic motor function, such as irritable bowel syndrome, are much more common. We have recently reported specialized basket-like structures, immunoreactive for calbindin, that likely underlie synaptic inputs to specific types of calretinin-immunoreactive neurons in the guinea-pig colon. Based on detailed immunohistochemical analysis, we postulated the recipient neurons may be excitatory motor neurons and ascending interneurons. In the present study, we combined retrograde tracing and immunohistochemistry to examine the projections of circular muscle motor neurons, myenteric interneurons, and putative sensory neurons. We focused on neurons with immunoreactivity for calbindin, calretinin and nitric oxide synthase and their relationship with calbindin baskets. Retrograde tracing using indocarbocyanine dye (DiI) revealed that many of the nerve cell bodies surrounded by calbindin baskets belong to motor neurons and ascending interneurons. Unique functional classes of myenteric neurons were identified based on morphology, neuronal markers and polarity of projection. We provide evidence for three groups of ascending motor neurons based on immunoreactivity and association with calbindin baskets, a finding that may have significant functional implications., (© 2018 Wiley Periodicals, Inc.)

Diabetic autonomic peripheral neuropathy (PN) involves a broad spectrum of organs. One of them is the gastrointestinal (GI) tract. The molecular mechanisms underlying the pathogenesis of digestive complications are not yet fully understood. Digestion is controlled by the central nervous system (CNS) and the enteric nervous system (ENS) within the wall of the GI tract. Enteric neurons exert regulatory effects due to the many biologically active substances secreted and released by enteric nervous system (ENS) structures. These include nitric oxide (NO), produced by the neural nitric oxide synthase enzyme (nNOS). It is a very important inhibitory factor, necessary for smooth muscle relaxation. Moreover, it was noted that nitrergic innervation can undergo adaptive changes during pathological processes. Additionally, nitrergic neurons function may be regulated through the synthesis of other active neuropeptides. Therefore, in the present study, using the immunofluorescence technique, we first examined the influence of hyperglycemia on the NOS- containing neurons in the porcine small intestine and secondly the co-localization of nNOS with vasoactive intestinal polypeptide (VIP), galanin (GAL) and substance P (SP) in all plexuses studied. Following chronic hyperglycaemia, we observed a reduction in the number of the NOS-positive neurons in all intestinal segments studied, as well as an increased in investigated substances in nNOS positive neurons. This observation confirmed that diabetic hyperglycaemia can cause changes in the neurochemical characteristics of enteric neurons, which can lead to numerous disturbances in gastrointestinal tract functions. Moreover, can be the basis of an elaboration of these peptides analogues utilized as therapeutic agents in the treatment of GI complications.

Damage of enteric neurons and partial or total loss of selective neuronal populations are reported in intestinal disorders including inflammatory bowel diseases and necrotizing enterocolitis. To develop three-dimensional scaffolds for enteric neurons we propose the decoration of ionic-complementary self-assembling peptide (SAP) hydrogels, namely EAK or EAbuK, with bioactive motives. Our results showed the ability of EAK in supporting neuronal cell attachment and neurite development. Therefore, EAK was covalently conjugated to: RGD, (GRGDSP) 4 K (fibronectin), FRHRNRKGY (h-vitronectin, named HVP), IKVAV (laminin), and type 1 Insulin-like Growth Factor (IGF-1). Chemoselective ligation was applied for the SAP conjugation with IGF-1 and the other longer sequences. Freshly isolated murine enteric neurons attached and grew on all functionalized EAK but IGF-1. Cell-cell contact was evident on hydrogels enriched with (GRGDSP) 4 K and HVP. Moreover (GRGDSP) 4 K significantly increased mRNA expression of neurotrophin-3 and nerve growth factor, two trophic factors supporting neuronal survival and differentiation, whereas IKVAV decoration specifically increased mRNA expression of acetylcholinesterase and choline acetyltransferase, genes involved in synaptic communication between cholinergic neurons. Thus, decorated hydrogels are proposed as injectable scaffolds to support in loco survival of enteric neurons, foster synaptic communication, or drive the differentiation of neuronal subtypes.

Diabetes mellitus (DM) may lead to gastrointestinal motility disorders. Rodent models of DM indicate the presence of morpho-functional abnormalities of the enteric nervous system. Here, we evaluated whether experimental DM can cause changes in the excitatory cholinergic fibers, neuronal density, and voltage-gated sodium channel (Nav) expression in the myenteric plexus of the ileum. After streptozotocin-induced hyperglycemia in female rats progressed for eight weeks, triple immunofluorescence labeling experiments revealed that the neuronal density in DM rats was significantly lower than that in control. On average, the density of total neurons reduced by 52.2% (p = 0.0001), cholinergic neurons by 50.0% (p = 0.0068), and nitrergic neurons by 54.8% (p = 0.0042). The number of neurons per ganglionic area was also significantly reduced (to 28.2% of total neurons, p = 0.0002; 27.7% of cholinergic neurons, p = 0.0002, and 32.1% of nitrergic neurons, p = 0.0016). Furthermore, the density of the cholinergic fibers at the surface of the longitudinal muscle was significantly reduced (DM: 24 ± 3%; p = 0.003, control: 41 ± 2%); however, western-blot analysis did not indicate a reduction in the expression of choline acetyltransferase (ChAT) in the DM group. The Nav1.6 isoform was detected in different myenteric neurons of the ileum. RT-qPCR data did not suggest an alteration of transcripts for ChAT, neuronal nitric oxide synthase, Nav1.3, Nav1.6, or Nav1.7. Our data support the view that chronic DM leads to a reduction of excitatory cholinergic fibers and neuronal density. However, changes in sodium channel expression pattern, which could cause neuronal dysfunction, were not detected., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Alpha-synuclein (α-Syn) immunostaining in the enteric nervous system (ENS) has been investigated to determine the role of diagnostic biomarker of Parkinson's disease (PD). However, determining factors for alpha-synuclein (α-Syn) deposition in the ENS of humans are still unclear. We aimed to investigate a possible association between SNCA variants and the presence of α-Syn immunostaining in the ENS in patients with PD and healthy individuals., Methods: The study subjects consisted of 38 patients with PD and 46 healthy individuals. α-Syn immunohistochemistry was performed for gastric and colonic mucosal tissues of patients with PD and controls. Mucosal biopsy tissues of the ENS were obtained using standard biopsy forceps by endoscopic gastroduodenoscopy or colonoscopy. Two variants within the SNCA gene (the single nucleotide polymorphism [SNP] rs11931074 and the microsatellite REP1) were genotyped., Results: In patients with PD, the rs11931074 (G allele) was significantly associated with the presence of α-Syn immunostaining in the ENS (OR = 5.96, 95% CI = 1.70-20.97, P = 0.01). In an interaction analysis, SNP rs11931074-PD status interaction was significantly associated with positive α-Syn immunostaining in the ENS (OR = 7.33, 95% CI = 1.58-33.88, P = 0.01). Longer SNCA REP1 alleles were not associated with positive α-Syn immunostaining in the ENS., Conclusion: This exploratory study demonstrated that α-Syn deposition in the ENS may be associated with SNCA variants in patients with PD., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Introduction: Dysautonomia in Parkinson's disease (PD) has been shown to be associated with disease severity and especially with the occurrence of dementia. One proposed explanation for this finding is that phosphorylated alpha-synuclein histopathology (PASH), the characteristic pathological feature of PD is more diffuse in dysautonomia-associated PD than in disease without dysautonomia, not only in the central nervous system but also in peripheral autonomic networks. The aim of this study was therefore to determine if colonic alpha-synuclein histopathology is associated with dysautonomia in PD., Methods: A total of 43 PD patients participated in this study. For each patient, two biopsies were taken in the sigmoid colon and analyzed by immunohistochemistry with antibodies against phosphorylated alpha-synuclein and PGP 9.5. All patients had a complete neuropsychological and neurological assessment along with a comprehensive evaluation of dysautonomia with questionnaires (SCOPA-Aut, NMS-Quest, Rome III constipation criteria and dry eye symptoms) and functional tests (pupillometry, Saxon and Schirmer's tests, heart rate variability, orthostatic blood pressure measure and sympathetic skin response)., Results: Colonic PASH was observed in 20/43 PD patients. No differences were observed in autonomic symptoms and testing between patients with and without PASH., Conclusions: Although frequent in PD, autonomic dysfunction is not related to colonic PASH. In addition to the existing literature, our findings further suggest that each dysautonomic symptom in PD might not be associated with a more severe or diffuse PASH not only in the central nervous system but also in the peripheral autonomic nervous systems., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The epithelial lining of the gastrointestinal (GI) tract in conjunction with the enteric nervous system (ENS) plays an important role in mediating solute absorption and secretion. A dysregulated ionic movement across the epithelium can result in GI diseases that manifest as either watery diarrhea or constipation. Hirschsprung disease is an example of an ENS disorder characterized by absence of enteric ganglia in distal gut resulting in obstructive phenotype. Receptor rearranged during transfection (RET) gene variants are the most commonly recognized genetic associations with Hirschsprung disease. In this issue of Neurogastroenterology and Motility, Russell et al demonstrate that RET mediates colonic ion transport through modulation of cholinergic nerves. They go on to show inhibition of RET can attenuate accelerated transit in a rat model. Normalizing secretory and absorptive defects has been an attractive therapeutic strategy. In addition to the intrinsic regulation of secretory processes, luminal mediators like bile acids, short-chain fatty acids, and proteases can affect both secretion and barrier function of the intestinal epithelium. Elevated levels of proteases have been identified in a wide range of GI diseases including irritable bowel syndrome. Proteases are known to cause visceral hypersensitivity and barrier disruption in vitro and in animal models. The goals of this review are to describe fundamental concepts related to intestinal epithelial secretion, the utility of Ussing chambers to measure ionic mechanisms and to discuss examples of novel signaling pathways; namely the RET signaling cascade in secretomotor neurons and effects of luminal proteases on barrier and ionic secretion., (© 2019 John Wiley & Sons Ltd.)

Recently, we reported that non-sulfated cholecystokinin-8 (NS CCK-8) reduces food intake by cholecystokinin-B receptors (CCK-BR). To examine a possible site of action for this peptide, and based on the fact that both NS CCK-8 and CCK-BR are found centrally and peripherally, in the current study we hypothesized that NS CCK-8 increases Fos-like immunoreactivity (Fos-LI, a neuronal activation marker) in the dorsal vagal complex (DVC) of the hindbrain and the myenteric and submucosal plexuses of the small intestine. We found that intraperitoneal NS CCK-8 (0.5 nmol/kg) increases Fos-LI in the DVC, the myenteric and the submucosal plexuses of the duodenum and the myenteric plexus of the jejunum. The findings suggest, but does not prove, a potential role for the DVC and the enteric neurons in the feeding responses evoked by NS CCK-8., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: The expression of RET in the developing enteric nervous system (ENS) suggests that RET may contribute to adult intestinal function. ENS cholinergic nerves play a critical role in the control of colonic function through the release of acetylcholine (ACh). In the current study, we hypothesized that a RET-mediated mechanism may regulate colonic ion transport and motility through modulation of cholinergic nerves., Methods: The effect of RET inhibition on active ion transport was assessed electrophysiologically in rat colonic tissue mounted in Ussing chambers via measurements of short circuit current (Isc) upon electrical field stimulation (EFS) or pharmacologically with cholinergic agonists utilizing a gastrointestinal (GI)-restricted RET inhibitor. We assessed the effect of the RET inhibitor on propulsive motility via quantification of fecal pellet output (FPO) induced by the acetylcholinesterase inhibitor neostigmine., Key Results: We found that enteric ganglia co-expressed RET and choline acetyltransferase (ChAT) transcripts. In vitro, the RET kinase inhibitor GSK3179106 attenuated the mean increase in Isc induced by either EFS or carbachol but not bethanechol. In vivo, GSK3179106 significantly reduced the prokinetic effect of neostigmine., Conclusion and Inferences: Our findings provide evidence that RET-mediated mechanisms regulate colonic function by maintaining cholinergic neuronal function and enabling ACh-evoked chloride secretion and motility. We suggest that modulating the cholinergic control of the colon via a RET inhibitor may represent a novel target for the treatment of intestinal disorders associated with increased secretion and accelerated GI transit such as irritable bowel syndrome with diarrhea (IBS-D)., (© 2018 John Wiley & Sons Ltd.)

The enteric nervous system (ENS) represents a vast network of neuronal and glial cell types that develops entirely from migratory neural crest (NC) progenitor cells. Considerable improvements in the understanding of the molecular mechanisms underlying NC induction and regional specification have recently led to the development of a robust method to re-create the process in vitro using human pluripotent stem cells (hPSCs). Directing the fate of hPSCs toward the enteric NC (ENC) results in an accessible and scalable in vitro model of ENS development. The application of hPSC-derived enteric neural lineages provides a powerful platform for ENS-related disease modeling and drug discovery. Here we present a detailed protocol for the induction of a regionally specific NC intermediate that occurs over the course of a 15-d interval and is an effective source for the in vitro derivation of functional enteric neurons (ENs) from hPSCs. Additionally, we introduce a new and improved protocol that we have developed to optimize the protocol for future applications in regenerative medicine, in which components of undefined activity have been replaced with fully defined culture conditions. This protocol provides access to a broad range of human ENS lineages within a 30-d period.

Background: Diverticular disease, a major gastrointestinal disorder, is associated with modifications of the enteric nervous system, encompassing alterations of neurochemical coding and of the tyrosine receptor kinase Ret/GDNF pathway. However, molecular factors underlying these changes remain to be determined., Objectives: We aimed to characterise the expression of Phox2b, an essential regulator of Ret and of neuronal subtype development, in the adult human enteric nervous system, and to evaluate its potential involvement in acute diverticulitis., Methods: Site-specific gene expression of Phox2b in the adult colon was analysed by quantitative polymerase chain reaction. Colonic specimens of adult controls and patients with diverticulitis were subjected to quantitative polymerase chain reaction for Phox2b and dual-label immunochemistry for Phox2b and the neuronal markers RET and tyrosine hydroxylase or the glial marker S100β., Results: The results indicate that Phox2b is physiologically expressed in myenteric neuronal and glial subpopulations in the adult enteric nervous system. Messenger RNA expression of Phox2b was increased in patients with diverticulitis and both neuronal, and glial protein expression of Phox2b were altered in these patients., Conclusions: Alterations of Phox2b expression may contribute to the enteric neuropathy observed in diverticular disease. Future studies are required to characterise the functions of Phox2b in the adult enteric nervous system and to determine its potential as a therapeutic target in gastrointestinal disorders.

Piezo channels play fundamental roles in many physiological processes. Their presence and functional role in the enteric nervous system is still not known. We hypothesize that they play a role in mechanotransduction in enteric neurons. Our aims are to quantify the presence of both Piezo1 and 2 in enteric neurons throughout the gastrointestinal tract using immunohistochemistry and analyze their function(s) using neuroimaging techniques and pharmacological investigations. In order to perform a systematic and comparative study, we performed our experiments in gastrointestinal tissue from guinea pigs, mice and humans. Piezo1 (20-70%) is expressed by both enteric neuronal cell bodies and fibers in the myenteric and submucosal plexi of all the species investigated. Generally, Piezo1 expressing somata are more numerous in the submucosal plexus (50-80%) than in the myenteric plexus (15-35%) apart from the stomach where Piezo1 is expressed in up to 60% of cell bodies. Myenteric Piezo1 neurons mainly (60-100%) but not exclusively, also express nitric oxide synthase, a minority express choline acetyltransferase. In the submucosal plexus, Piezo1 neurons co-express vasoactive intestinal peptide (40-90%). Conversely, expression of Piezo2 is extremely rare in the somata of enteric neurons and is present in few neurites. In functional experiments, 38-76% of the mechanosensitive neurons expressed Piezo1 channels. Statistical analysis showed a positive significant correlation between mechanosensitive and Piezo1 positive neurons. However, pharmacological experiments using an activator and an inhibitor of Piezo channels did not demonstrate changes in mechanotransduction. A major role of Piezo1 in the mechanosensitivity of enteric neurons can be excluded.

The protein alpha-synuclein whose expression is strongly implicated in Parkinson's disease (PD) is not only expressed in the CNS but also in the enteric nervous system (ENS). The growing body of evidence suggesting that gastrointestinal inflammation is involved in the development of PD led us to investigate the effects of inflammation on alpha-synuclein expression in primary culture of rat ENS and in mice with dextran sulfate sodium-induced colitis. Using western blot and qPCR, we found that both lipopolysaccharide and a combination of tumor necrosis factor-α and interleukin 1-β decreased the expression levels of alpha-synuclein in primary culture of rat ENS, an effect that was prevented in the presence of the p38 inhibitors SB203580 and BIRB 796. Lipopolysaccharide and tumor necrosis factor-α/interleukin 1-β had no effect on alpha-synuclein expression in primary culture of rat CNS and in human erythroid leukemia cells. In mice, acute but not chronic dextran sulfate sodium-induced colitis was associated with a decreased expression of colonic alpha-synuclein. As a whole, our findings indicate that acute inflammatory insults down-regulate alpha-synuclein expression in the ENS via a p38 pathway. They provide new insights into the widely discussed concepts of alpha-synuclein expression and aggregation in the ENS in PD and raise issues about the possible role of gastrointestinal inflammation in the development of PD. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/., (© 2018 International Society for Neurochemistry.)

Antibiotic use during adolescence may result in dysbiosis-induced neuronal vulnerability both in the enteric nervous system (ENS) and central nervous system (CNS) contributing to the onset of chronic gastrointestinal disorders, such as irritable bowel syndrome (IBS), showing significant psychiatric comorbidity. Intestinal microbiota alterations during adolescence influence the expression of molecular factors involved in neuronal development in both the ENS and CNS. In this study, we have evaluated the expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tropomyosin-related kinase B (TrkB) in juvenile mice ENS and CNS, after a 2-week antibiotic (ABX) treatment. In both mucosa and mucosa-deprived whole-wall small intestine segments of ABX-treated animals, BDNF and TrKB mRNA and protein levels significantly increased. In longitudinal muscle-myenteric plexus preparations of ABX-treated mice the percentage of myenteric neurons staining for BDNF and TrkB was significantly higher than in controls. After ABX treatment, a consistent population of BDNF- and TrkB-immunoreactive neurons costained with SP and CGRP, suggesting up-regulation of BDNF signaling in both motor and sensory myenteric neurons. BDNF and TrkB protein levels were downregulated in the hippocampus and remained unchanged in the prefrontal cortex of ABX-treated animals. Immunostaining for BDNF and TrkB decreased in the hippocampus CA3 and dentate gyrus subregions, respectively, and remained unchanged in the prefrontal cortex. These data suggest that dysbiosis differentially influences the expression of BDNF-TrkB in the juvenile mice ENS and CNS. Such changes may potentially contribute later to the development of functional gut disorders, such as IBS, showing psychiatric comorbidity., Competing Interests: The authors have declared that no competing interests exist.

Background: Inflammatory bowel disease (IBD) is a functional disorder with chronic and relapsing clinical features. Vasopressin (VP) is a hormone responsible for water and stress homeostasis and also regulates gastrointestinal inflammation and motility. We explored whether VP was related to IBD pathogenesis and its possible pathway., Methods: Colitis was induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) in mice. The disease activity and colonic damage were evaluated through a scoring system. Locations of the V 1a receptor were revealed by immunochemistry method in colon. Ussing chamber technique was performed for the electrophysiological characterization by using rat ileum. The (Arg 8 )-Vasopressin (AVP)-evoked short-circuit current (Isc) was recorded in the presence of conivaptan (V 1a and V 2 receptor antagonist), tolvaptan (V 1b receptor antagonist), tetrodotoxin (TTX), atropine, cyclooxygenase (COX) inhibitors (indomethacin, nonspecific COX antagonist; SC560, COX-1 antagonist; NS560, COX-2 antagonist), and a stabilizer of mast cell (cromolyn sodium), respectively., Key Results: TNBS resulted in the obvious loss of body weight and tissue damages in mice. AVP significantly aggravated the TNBS-induced colitis, which was attenuated by conivaptan but not tolvaptan. V 1a receptors were found immunopositive in neurons among the enteric nervous system. AVP evoked a pulsatile response in Isc. Its amplitude, frequency, and cycle duration were around 8-15 µA/cm 2 , 10-11 mHz, and 1.5 minutes, respectively. Notably, the AVP-evoked change in Isc was abolished by TTX, atropine, conivaptan, indomethacin, NS560, and cromolyn sodium, respectively., Conclusions and Inferences: VP-V 1a receptor played the proinflammatory role in TNBS-induced colitis by promoting COX-2-dependent prostaglandin release from mucosal mast cells, which was mediated by the cholinergic pathway., (© 2018 John Wiley & Sons Ltd.)

Cells of the vagal neural crest (NC) form most of the enteric nervous system (ENS) by a colonising wave in the embryonic gut, with high cell proliferation and differentiation. Enteric neuropathies have an ENS deficit and cell replacement has been suggested as therapy. This would be performed post-natally, which raises the question of whether the ENS cell population retains its initial ENS-forming potential with age. We tested this on the avian model in organ culture in vitro (3 days) using recipient aneural chick midgut/hindgut combined with ENS-donor quail midgut or hindgut of ages QE5 to QE10. ENS cells from young donor tissues (≤ QE6) avidly colonised the aneural recipient, but this capacity dropped rapidly 2-3 days after the transit of the ENS cell wavefront. This loss in capability was autonomous to the ENS population since a similar decline was observed in ENS cells isolated by HNK1 FACS. Using QE5, 6, 8 and 10 midgut donors and extending the time of assay to 8 days in chorio-allantoic membrane grafts did not produce 'catch up' colonisation. NC-derived cells were counted in dissociated quail embryo gut and in transverse sections of chick embryo gut using NC, neuron and glial marker antibodies. This showed that the decline in ENS-forming ability correlated with a decrease in proportion of ENS cells lacking both neuronal and glial differentiation markers, but there were still large numbers of such cells even at stages with low colonisation ability. Moreover, ENS cells in small numbers from young donors were far superior in colonisation ability to larger numbers of apparently undifferentiated cells from older donors. This suggests that the decline of ENS-forming ability has both quantitative and qualitative aspects. In this case, ENS cells for cell therapies should aim to replicate the embryonic ENS stage rather than using post-natal ENS stem/progenitor cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The enteric nervous system is mostly derived from vagal neural crest (NC) cells adjacent to somites (s)1-7. We used in ovo focal fluorescent vital dyes and focal electroporation of fluorophore-encoding plasmids in quail embryos to investigate NC cell migration to the foregut initially and later throughout the entire gut. NC cells of different somite-level origins were largely separate until reaching the foregut at about QE2.5, when all routes converged. By QE3.5, NC cells of different somite-levels became mixed, although s1-s2 NC cells were mainly confined to rostral foregut. Mid-vagal NC-derived cells (s3 and s4 level) arrived earliest at the foregut, and occurred in greatest number. By QE6.5 ENS was present from foregut to hindgut. Mid-vagal NC-derived cells occurred in greatest numbers from foregut to distal hindgut. NC-derived cells of s2, s5, and s6 levels were fewer and were widely distributed but were never observed in the distal hindgut. Rostro-vagal (s1) and caudo-vagal (s7) levels were few and restricted to the foregut. Single somite levels of quail neural tube/NC from s1 to s8 were combined with chick aneural ChE4.5 midgut and hindgut and the ensemble was grown on the chorio-allantoic membrane for 6 days. This tests ENS-forming competence in the absence of intra-segmental competition between NC cells, of differential influences of segmental paraxial tissues, and of positional advantage. All vagal NC-levels, but not s8 level, furnished enteric plexuses in the recipient gut, but the density of both ENS cells in total and neurons was highest from mid-vagal level donors, as was the length colonised. We conclude that the fate and competence for ENS formation of vagal NC sub-levels is not uniform over the vagal level but is biased to favour mid-vagal levels. Overviewing this and prior studies suggests the vagal region is, as in its traditional sense, a natural unit but with complex sub-divisions., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background and Aims: Autism spectrum disorder (ASD) is currently estimated to affect more than 1% of the world population. For people with ASD, gastrointestinal (GI) distress is a commonly reported but a poorly understood co-occurring symptom. Here, we investigate the physiological basis for GI distress in ASD by studying gut function in a zebrafish model of Phelan-McDermid syndrome (PMS), a condition caused by mutations in the SHANK3 gene., Methods: To generate a zebrafish model of PMS, we used CRISPR/Cas9 to introduce clinically related C-terminal frameshift mutations in shank3a and shank3b zebrafish paralogues ( shank3abΔC ). Because PMS is caused by SHANK3 haploinsufficiency, we assessed the digestive tract (DT) structure and function in zebrafish shank3abΔC +/- heterozygotes. Human SHANK3 mRNA was then used to rescue DT phenotypes in larval zebrafish., Results: Significantly slower rates of DT peristaltic contractions ( p  < 0.001) with correspondingly prolonged passage time ( p  < 0.004) occurred in shank3abΔC +/- mutants. Rescue injections of mRNA encoding the longest human SHANK3 isoform into shank3abΔC +/- mutants produced larvae with intestinal bulb emptying similar to wild type (WT), but still deficits in posterior intestinal motility. Serotonin-positive enteroendocrine cells (EECs) were significantly reduced in both shank3abΔC +/- and shank3abΔC -/- mutants ( p  < 0.05) while enteric neuron counts and overall structure of the DT epithelium, including goblet cell number, were unaffected in shank3abΔC +/- larvae., Conclusions: Our data and rescue experiments support mutations in SHANK3 as causal for GI transit and motility abnormalities. Reductions in serotonin-positive EECs and serotonin-filled ENS boutons suggest an endocrine/neural component to this dysmotility. This is the first study to date demonstrating DT dysmotility in a zebrafish single gene mutant model of ASD., Competing Interests: All procedures with animal are described in protocols 15–191 and 18–128 and were approved by the Institutional Animal Care and Use Committee of University of Miami.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

The enteric nervous system is thought to originate solely from the neural crest. Transgenic lineage tracing revealed a novel population of clonal pancreatic duodenal homeobox-1 (Pdx1)-Cre lineage progenitor cells in the tunica muscularis of the gut that produced pancreatic descendants as well as neurons upon differentiation in vitro. Additionally, an in vivo subpopulation of endoderm lineage enteric neurons, but not glial cells, was seen especially in the proximal gut. Analysis of early transgenic embryos revealed Pdx1-Cre progeny (as well as Sox-17-Cre and Foxa2-Cre progeny) migrating from the developing pancreas and duodenum at E11.5 and contributing to the enteric nervous system. These results show that the mammalian enteric nervous system arises from both the neural crest and the endoderm. Moreover, in adult mice there are separate Wnt1-Cre neural crest stem cells and Pdx1-Cre pancreatic progenitors within the muscle layer of the gut., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)