Mesenchymal cells regulate enteric neural crest cell migration via RET-GFRA1b trans-signaling.

During early development, the enteric nervous system forms from the migration of enteric neural crest cells (ENCCs) from the foregut to the hindgut, where they undergo proliferation and differentiation facilitated by interactions with enteric mesenchymal cells (EMCs). This study investigates the impact on ENCC migration of EMC-ENCC communication mediated by GFRA1b expressed in EMCs. GFRA1-expressing cells in day 11-12 (E11-12) mouse embryos differentiated into smooth muscle cells from E12 onwards. Observations at E12-13.5 revealed high levels of GFRA1 expression on the anti-mesenteric side of the hindgut, correlating with enhanced ENCC migration. This indicates that GFRA1 in EMCs plays a role in ENCC migration during development. Examining GFRA1 isoforms, we found high levels of GFRA1b, which lacks amino acids 140-144, in EMCs. To assess the impact of GFRA1 isoforms on EMC-ENCC communication, we conducted neurosphere drop assays. This revealed that GFRA1b-expressing cells promoted GDNF-dependent extension and increased neurite density in ENCC neurospheres. Co-culture of ENCC mimetic cells expressing RET and GFRA1a with EMC mimetic cells expressing GFRA1a, GFRA1b, or vector alone showed that only GFRA1b-expressing co-cultured cells sustained RET phosphorylation in ENCC-mimetic cells for over 120 min upon GDNF stimulation. Our study provides evidence that GFRA1b-mediated cell-to-cell communication plays a critical role in ENCC motility in enteric nervous system development. These findings contribute to understanding the cellular interactions and signaling mechanisms that underlie enteric nervous system formation and highlight potential therapeutic targets for gastrointestinal motility disorders.

The role of galanin in the progression and prognosis of colorectal cancer: the unfinished story.

The paper presents a summary of immunohistochemical (IHC) and biochemical investigations on the presence of galanin (Gal), one of the neuropeptides abundant in the enteric nervous systems, and three types of its receptors (GalR1-3) in colorectal cancer (CRC) tissue and non-involved colon wall and their associations with clinical-pathological data of the CRC patients. We were the first to morphologically demonstrate the presence of endogenous Gal in CRC sections and measure its content in homogenates of tumor tissue and dissected compartments of unchanged colon wall. The prominent atrophy of myenteric plexuses displaying Gal immunoreactivity (Gal-Ir) located close to the tumor invasion was found to be accompanied by higher Gal content in the tumor-adjacent muscularis externa than in tumor-distant tissue. In further studies for the first time, we demonstrated by the IHC technique the presence of the GalR1-3 receptors in the CRC tumors and the colon mucosa and found that higher GalR3-Ir in the tumor tissue correlated with longer overall survival of CRC patients. Furthermore, we discovered that lower GalR1 expression in submucosal plexuses located near the tumor correlated with a better prognosis in patients with CRC. These findings suggest that GalR1 could be considered as a novel therapeutic target in CRC. In conclusion, our morphological investigations provided novel data documenting the involvement of Gal and its receptors in the progression of CRC and showed the usefulness of the IHC technique for the prognosis of CRC patients.

Autologous cell transplantation for treatment of colorectal aganglionosis in mice.

Neurointestinal diseases cause significant morbidity and effective treatments are lacking. This study aimes to test the feasibility of transplanting autologous enteric neural stem cells (ENSCs) to rescue the enteric nervous system (ENS) in a model of colonic aganglionosis. ENSCs are isolated from a segment of small intestine from Wnt1::Cre;R26iDTR mice in which focal colonic aganglionosis is simultaneously created by diphtheria toxin injection. Autologous ENSCs are isolated, expanded, labeled with lentiviral-GFP, and transplanted into the aganglionic segment in vivo. ENSCs differentiate into neurons and glia, cluster to form neo-ganglia, and restore colonic contractile activity as shown by electrical field stimulation and optogenetics. Using a non-lethal model of colonic aganglionosis, our results demonstrate the potential of autologous ENSC therapy to improve functional outcomes in neurointestinal disease, laying the groundwork for clinical application of this regenerative cell-based approach.

Phosphatase and Tensin Homolog Inhibition in Proteolipid Protein 1-Expressing Cells Stimulates Neurogenesis and Gliogenesis in the Postnatal Enteric Nervous System.

Phosphatase and tensin homolog (Pten) is a key regulator of cell proliferation and a potential target to stimulate postnatal enteric neuro- and/or gliogenesis. To investigate this, we generated two tamoxifen-inducible Cre recombinase murine models in which Pten was conditionally ablated, (1) in glia (Plp1-expressing cells) and (2) in neurons (Calb2-expressing cells). Tamoxifen-treated adult (7-12 weeks of age; n = 4-15) mice were given DSS to induce colitis, EdU to monitor cell proliferation, and were evaluated at two timepoints: (1) early (3-4 days post-DSS) and (2) late (3-4 weeks post-DSS). We investigated gut motility and evaluated the enteric nervous system. Pten inhibition in Plp1-expressing cells elicited gliogenesis at baseline and post-DSS (early and late) in the colon, and neurogenesis post-DSS late in the proximal colon. They also exhibited an increased frequency of colonic migrating motor complexes (CMMC) and slower whole gut transit times. Pten inhibition in Calb2-expressing cells did not induce enteric neuro- or gliogenesis, and no alterations were detected in CMMC or whole gut transit times when compared to the control at baseline or post-DSS (early and late). Our results merit further research into Pten modulation where increased glia and/or slower intestinal transit times are desired (e.g., short-bowel syndrome and rapid-transit disorders).

Pathophysiology and Management of Postoperative Ileus in Adults and Neonates: A Review.

Postoperative ileus (POI) is caused by enteric neural dysfunction and inflammatory response to the stress of surgery as well as the effect of anesthetics and opioid pain medications. POI results in prolonged hospital stays, increased medical costs, and diminished enteral nutrition, rendering it a problem worth tackling. Many cellular pathways are implicated in this disease process, creating numerous opportunities for targeted management strategies. There is a gap in the literature in studies exploring neonatal POI pathophysiology and treatment options. It is well known that neonatal immune and enteric nervous systems are immature, and this results in gut physiology which is distinct from adults. Neonates undergoing abdominal surgery face similar surgical stressors and exposure to medications that cause POI in adults. In this review, we aim to summarize the existing adult and neonatal literature on POI pathophysiology and management and explore applications in the neonatal population.

The critical role of muscularis macrophages in modulating the enteric nervous system function and gastrointestinal motility.

Macrophages are the originators of inflammatory compounds, phagocytic purifiers in their local environment, and wound healing protectors in oxidative environments. They are molded by the tissue milieu they inhabit, with gastrointestinal (GI) muscularis macrophages (MMs) being a prime example. MMs are located in the muscular layer of the GI tract and contribute to muscle repair and maintenance of GI motility. MMs are often in close proximity to the enteric nervous system, specifically near the enteric neurons and interstitial cells of Cajal (ICCs). Consequently, the anti-inflammatory function of MMs corresponds to the development and maintenance of neural networks in the GI tract. The capacity of MMs to shift from anti-inflammatory to proinflammatory states may contribute to the inflammatory aspects of various GI diseases and disorders such as diabetic gastroparesis or postoperative ileus, functional disorders such as irritable bowel syndrome, and organic diseases such as inflammatory bowel disease. We reviewed the current knowledge of MMs and their influence on neighboring cells due to their important role in the GI tract.

Spatial gene expression profile of Wnt-signaling components in the murine enteric nervous system.

Introduction: Wnt-signaling is a key regulator of stem cell homeostasis, extensively studied in the intestinal crypt and other metazoan tissues. Yet, there is hardly any data available on the presence of Wnt-signaling components in the adult enteric nervous system (ENS) in vivo. Methods: Therefore, we employed RNAscope HiPlex-assay, a novel and more sensitive in situ hybridization technology. By amplifying target specific signals, this technique enables the detection of low abundance, tightly regulated RNA content as is the case for Wnt-signaling components. Additionally, we compared our data to previously published physiological single cell RNA and RiboTag-based RNA sequencing analyses of enteric gliosis using data-mining approaches. Results: Our descriptive analysis shows that several components of the multidi-mensional regulatory network of the Wnt-signaling pathway are present in the murine ENS. The transport and secretion protein for Wnt-ligands Wntless as well as canonical (Wnt3a and Wnt2b) and non-canonical Wnt-ligands (Wnt5a, Wnt7a, Wnt8b and Wnt11) are detectable within submucosal and myenteric plexus. Further, corresponding Frizzled receptors (Fzd1, Fzd3, Fzd6, and Fzd7) and regulatory signaling mediators like R-Spondin/DKK ligands are present in the ENS of the small and large intestine. Further, data mining approaches revealed, that several Wnt-related molecules are expressed by enteric glial cell clusters and are dynamically regulated during the inflammatory manifestation of enteric gliosis. Discussion: Our results suggest, that canonical and non-canonical Wnt-signaling has a much broader impact on the mature ENS and its cellular homeostasis in health and inflammation, than previously anticipated.

Updates and Challenges in ENS Cell Therapy for the Treatment of Neurointestinal Diseases.

Neurointestinal diseases represent a significant challenge in clinical management with current palliative approaches failing to overcome disease and treatment-related morbidity. The recent progress with cell therapy to restore missing or defective components of the gut neuromusculature offers new hope for potential cures. This review discusses the progress that has been made in the sourcing of putative stem cells and the studies into their biology and therapeutic potential. We also explore some of the practical challenges that must be overcome before cell-based therapies can be applied in the clinical setting. Although a number of obstacles remain, the rapid advances made in the enteric neural stem cell field suggest that such therapies are on the near horizon.

Differences in enteric neuronal density in the NSE-Noggin mouse model across institutes.

The enteric nervous system (ENS) is a large and complex part of the peripheral nervous system, and it is vital for gut homeostasis. To study the ENS, different hyper- and hypo-innervated model systems have been developed. The NSE-Noggin mouse model was described as one of the few models with a higher enteric neuronal density in the colon. However, in our hands NSE-Noggin mice did not present with a hyperganglionic phenotype. NSE-Noggin mice were phenotyped based on fur appearance, genotyped and DNA sequenced to demonstrate transgene and intact NSE-Noggin-IRES-EGFP construct presence, and RNA expression of Noggin was shown to be upregulated. Positive EGFP staining in the plexus of NSE-Noggin mice also confirmed Noggin protein expression. Myenteric plexus preparations of the colon were examined to quantify both the overall density of enteric neurons and the proportions of enteric neurons expressing specific subtype markers. The total number of enteric neurons in the colonic myenteric plexus of transgenic mice did not differ significantly from wild types, nor did the proportion of calbindin, calretinin, or serotonin immunoreactive myenteric neurons. Possible reasons as to why the hyperinnervated phenotype could not be observed in contrast with original studies using this mouse model are discussed, including study design, influence of microbiota, and other environmental variables.

Characterization of mitochondria-associated ER membranes in the enteric nervous system under physiological and pathological conditions.

Alterations in endoplasmic reticulum (ER)-mitochondria associations and in mitochondria-associated ER membrane (MAM) behavior have been reported in the brain in several neurodegenerative diseases. Despite the emerging role of the gut-brain axis in neurodegenerative disorders, the biology of MAM in the enteric nervous system (ENS) has not previously been studied. Therefore, we set out to characterize the MAM in the distal colon of wild-type C57BL/6J mice and senescence-accelerated mouse prone 8 (SAMP8), a mouse model of age-related neurodegeneration. We showed for the first time that MAMs are widely present in enteric neurons and that their association is altered in SAMP8 mice. We then examined the functions of MAMs in a primary culture model of enteric neurons and showed that calcium homeostasis was altered in SAMP8 mice when compared with control animals. These findings provide the first detailed characterization of MAMs in the ENS under physiological conditions and during age-associated neurodegeneration. Further investigation of MAM modifications in the ENS in disease may provide valuable information about the possible role of enteric MAMs in neurodegenerative diseases.NEW & NOTEWORTHY Our work shows for the first time the presence of contacts between endoplasmic reticulum and mitochondria in the enteric neurons and that the dynamic of these contacts is affected in these cells from an age-related neurodegeneration mouse model. It provides new insights into the potential role of enteric mitochondria-associated endoplasmic reticulum membrane in neurodegenerative disorders.

EphB2 promotes enteric nitrergic hyperinnervation and neurogenic inflammation in DSS-induced chronic colitis in mice.

BACKGROUND: Enteric nervous system (ENS) has been closely associated with the neuro-immune response and is currently considered a reliable target for intestinal inflammation. Neuronal nitric oxide synthase (nNOS) nerves are involved in inflammatory diseases by releasing nitric oxide (NO). EphB2 expression and density of innervation of the mucosal layer are positively correlated with the severity of intestinal inflammatory responses. In this study, we hypothesized that a EphB2-mediated mechanism may regulate enteric immunity through modulation of nNOS nerves. METHODS: Firstly, the Western blot (WB) method was employed to quantify EphB2 expression in the intestinal mucosal layer of DSS mice and assess alterations in nerve fiber activation and density. Immunofluorescence (IF) double staining with nNOS and neuronal marker PGP9.5 was conducted to measure nNOS nerve fiber density within the intestinal mucosal layer of mice. Subsequently, in vivo experiments were performed to investigate the inhibitory or activatory effect of EphB2Fc or EphrinB2Fc on EphB2 expression and activation. Immunoprecipitation experiments confirmed the interaction between EphB2 and nNOS nerves. WB and IF experiments were carried out to evaluate both inflammatory conditions of mouse colonic mucosa following intervention with EphB2Fc/EphrinB2Fc as well as changes in nNOS nerve fibers expression. Finally, in vitro experiments, neurally-mediated inflammation was assessed in the organ bath system by activating intestinal mucosal innervation through Veratridine (VER) and electrical field stimulation (EFS) techniques for 3 h. The activation of nNOS nerves was inhibited by nitroindazole (7NI). WB was employed to detect changes in the expression of inflammatory factors in the intestinal mucosal layer in EphB2Fc/EphrinB2Fc treated mice and control group. KEY RESULTS: We found that the expression of EphB2 and density nNOS nerve fibers in the intestinal mucosa were positively correlated with the colitis response. Blocking (EphB2Fc)/activating (EphrinB2Fc) EphB2 in vivo significantly reduced/increased the density of nNOS nerve fibers and expression of inflammatory factors in colonic mucosa of DSS treated mice. In vitro, blocking nNOS nerves activation attenuated the inflammatory reaction induced by either EFS or EphB2. CONCLUSIONS: Our findings provided evidence that EphB2 mediated regulation of innate immunity-ENS crosstalk might represent an attractive target for novel therapeutic strategies in ulcerative colitis.

Improving morphological and functional properties of enteric neuronal networks in vitro using a novel upside-down culture approach.

The enteric nervous system (ENS) comprises millions of neurons and glia embedded in the wall of the gastrointestinal tract. It not only controls important functions of the gut but also interacts with the immune system, gut microbiota, and the gut-brain axis, thereby playing a key role in the health and disease of the whole organism. Any disturbance of this intricate system is mirrored in an alteration of electrical functionality, making electrophysiological methods important tools for investigating ENS-related disorders. Microelectrode arrays (MEAs) provide an appropriate noninvasive approach to recording signals from multiple neurons or whole networks simultaneously. However, studying isolated cells of the ENS can be challenging, considering the limited time that these cells can be kept vital in vitro. Therefore, we developed an alternative approach cultivating cells on glass samples with spacers (fabricated by photolithography methods). The spacers allow the cells to grow upside down in a spatially confined environment while enabling acute consecutive recordings of multiple ENS cultures on the same MEA. Upside-down culture also shows beneficial effects on the growth and behavior of enteric neural cultures. The number of dead cells was significantly decreased, and neural networks showed a higher resemblance to the myenteric plexus ex vivo while producing more stable signals than cultures grown in the conventional way. Overall, our results indicate that the upside-down approach not only allows to investigate the impact of neurological diseases in vitro but could also offer insights into the growth and development of the ENS under conditions much closer to the in vivo environment.NEW & NOTEWORTHY In this study, we devised a novel approach for culturing and electrophysiological recording of the enteric nervous system using custom-made glass substrates with spacers. This allows to turn cultures of isolated myenteric plexus upside down, enhancing the use of the microelectrode array technique by allowing recording of multiple cultures consecutively using only one chip. In addition, upside-down culture led to significant improvements in the culture conditions, resulting in a more in vivo-like growth.

Gastrointestinal dysmotility in rodent models of Parkinson's disease.

Multiple studies describe prodromal, nonmotor dysfunctions that affect the quality of life of patients who subsequently develop Parkinson's disease (PD). These prodromal dysfunctions comprise a wide array of autonomic issues, including severe gastrointestinal (GI) motility disorders such as dysphagia, delayed gastric emptying, and chronic constipation. Indeed, strong evidence from studies in humans and animal models suggests that the GI tract and its neural, mainly vagal, connection to the central nervous system (CNS) could have a major role in the etiology of PD. In fact, misfolded α-synuclein aggregates that form Lewy bodies and neurites, i.e., the histological hallmarks of PD, are detected in the enteric nervous system (ENS) before clinical diagnosis of PD. The aim of the present review is to provide novel insights into the pathogenesis of GI dysmotility in PD, focusing our attention on functional, neurochemical, and molecular alterations in animal models.

Molecular Mechanisms of Reelin in the Enteric Nervous System and the Microbiota-Gut-Brain Axis: Implications for Depression and Antidepressant Therapy.

Current pharmacological treatments for depression fail to produce adequate remission in a significant proportion of patients. Increasingly, other systems, such as the microbiome-gut-brain axis, are being looked at as putative novel avenues for depression treatment. Dysbiosis and dysregulation along this axis are highly comorbid with the severity of depression symptoms. The endogenous extracellular matrix protein reelin is present in all intestinal layers as well as in myenteric and submucosal ganglia, and its receptors are also present in the gut. Reelin secretion from subepithelial myofibroblasts regulates cellular migration along the crypt-villus axis in the small intestine and colon. Reelin brain expression is downregulated in mood and psychotic disorders, and reelin injections have fast antidepressant-like effects in animal models of depression. This review seeks to discuss the roles of reelin in the gastrointestinal system and propose a putative role for reelin actions in the microbiota-gut-brain axis in the pathogenesis and treatment of depression, primarily reflecting on alterations in gut epithelial cell renewal and in the clustering of serotonin transporters.

Stroke Alters the Function of Enteric Neurons to Impair Smooth Muscle Relaxation and Dysregulates Gut Transit.

BACKGROUND: Gut dysmotility is common after ischemic stroke, but the mechanism underlying this response is unknown. Under homeostasis, gut motility is regulated by the neurons of the enteric nervous system that control contractile/relaxation activity of muscle cells in the gut wall. More recently, studies of gut inflammation revealed interactions of macrophages with enteric neurons are also involved in modulating gut motility. However, whether poststroke gut dysmotility is mediated by direct signaling to the enteric nervous system or indirectly via inflammatory macrophages is unknown. METHODS AND RESULTS: We examined these hypotheses by using a clinically relevant permanent intraluminal midcerebral artery occlusion experimental model of stroke. At 24 hours after stroke, we performed in vivo and ex vivo gut motility assays, flow cytometry, immunofluorescence, and transcriptomic analysis. Stroke-induced gut dysmotility was associated with recruitment of muscularis macrophages into the gastrointestinal tract and redistribution of muscularis macrophages away from myenteric ganglia. The permanent intraluminal midcerebral artery occlusion model caused changes in gene expression in muscularis macrophages consistent with an altered phenotype. While the size of myenteric ganglia after stroke was not altered, myenteric neurons from post-permanent intraluminal midcerebral artery occlusion mice showed a reduction in neuronal nitric oxide synthase expression, and this response was associated with enhanced intestinal smooth muscle contraction ex vivo. Finally, chemical sympathectomy with 6-hydroxydopamine prevented the loss of myenteric neuronal nitric oxide synthase expression and stroke-induced slowed gut transit. CONCLUSIONS: Our findings demonstrate that activation of the sympathetic nervous system after stroke is associated with reduced neuronal nitric oxide synthase expression in myenteric neurons, resulting in impaired smooth muscle relaxation and dysregulation of gut transit.

Genetic regulation of enteric nervous system development in zebrafish.

The enteric nervous system (ENS) is a complex series of interconnected neurons and glia that reside within and along the entire length of the gastrointestinal tract. ENS functions are vital to gut homeostasis and digestion, including local control of peristalsis, water balance, and intestinal cell barrier function. How the ENS develops during embryological development is a topic of great concern, as defects in ENS development can result in various diseases, the most common being Hirschsprung disease, in which variable regions of the infant gut lack ENS, with the distal colon most affected. Deciphering how the ENS forms from its progenitor cells, enteric neural crest cells, is an active area of research across various animal models. The vertebrate animal model, zebrafish, has been increasingly leveraged to understand early ENS formation, and over the past 20 years has contributed to our knowledge of the genetic regulation that underlies enteric development. In this review, I summarize our knowledge regarding the genetic regulation of zebrafish enteric neuronal development, and based on the most current literature, present a gene regulatory network inferred to underlie its construction. I also provide perspectives on areas for future zebrafish ENS research.

From the Gut to the Brain: The Role of Enteric Glial Cells and Their Involvement in the Pathogenesis of Parkinson's Disease.

The brain-gut axis has been identified as an important contributor to the physiopathology of Parkinson's disease. In this pathology, inflammation is thought to be driven by the damage caused by aggregation of α-synuclein in the brain. Interestingly, the Braak's theory proposes that α-synuclein misfolding may originate in the gut and spread in a "prion-like" manner through the vagus nerve into the central nervous system. In the enteric nervous system, enteric glial cells are the most abundant cellular component. Several studies have evaluated their role in Parkinson's disease. Using samples obtained from patients, cell cultures, or animal models, the studies with specific antibodies to label enteric glial cells (GFAP, Sox-10, and S100ß) seem to indicate that activation and reactive gliosis are associated to the neurodegeneration produced by Parkinson's disease in the enteric nervous system. Of interest, Toll-like receptors, which are expressed on enteric glial cells, participate in the triggering of immune/inflammatory responses, in the maintenance of intestinal barrier integrity and in the configuration of gut microbiota; thus, these receptors might contribute to Parkinson's disease. External factors like stress also seem to be relevant in its pathogenesis. Some authors have studied ways to reverse changes in EGCs with interventions such as administration of Tryptophan-2,3-dioxygenase inhibitors, nutraceuticals, or physical exercise. Some researchers point out that beyond being activated during the disease, enteric glial cells may contribute to the development of synucleinopathies. Thus, it is still necessary to further study these cells and their role in Parkinson's disease.

The Golgi complex of dopaminergic enteric neurons is fragmented in a hemiparkinsonian rat model.

Since gastrointestinal disorders are early consequences of Parkinson's disease (PD), this disease is clearly not restricted to the central nervous system (CNS), but also significantly affects the enteric nervous system (ENS). Large aggregates of the protein α-synuclein forming Lewy bodies, the prototypical cytopathological marker of this disease, have been observed in enteric nervous plexuses. However, their value in early prognosis is controversial. The Golgi complex (GC) of nigral neurons appears fragmented in Parkinson's disease, a characteristic common in most neurodegenerative diseases. In addition, the distribution and levels of regulatory proteins such as Rabs and SNAREs are altered, suggesting that PD is a membrane traffic-related pathology. Whether the GC of enteric dopaminergic neurons is affected by the disease has not yet been analyzed. In the present study, dopaminergic neurons in colon nervous plexuses behave as nigral neurons in a hemiparkinsonian rat model based on the injection of the toxin 6-OHDA. Their GCs are fragmented, and some regulatory proteins' distribution and expression levels are altered. The putative mechanisms of the transmission of the neurotoxin to the ENS are discussed. Our results support the possibility that GC structure and the level of some proteins, especially syntaxin 5, could be helpful as early indicators of the disease. RESEARCH HIGHLIGHTS: The Golgi complexes of enteric dopaminergic neurons appear fragmented in a Parkinson's disease rat model. Our results support the hypothesis that the Golgi complex structure and levels of Rab1 and syntaxin 5 could be helpful as early indicators of the disease.

Comparison of wholemount dissection methods for neuronal subtype marker expression in the mouse myenteric plexus.

BACKGROUND: Accurately reporting the identity and representation of enteric nervous system (ENS) neuronal subtypes along the length of the gastrointestinal (GI) tract is critical to advancing our understanding of ENS control of GI function. Reports of varying proportions of subtype marker expression have employed different dissection techniques to achieve wholemount muscularis preparations of myenteric plexus. In this study, we asked whether differences in GI dissection methods could introduce variability into the quantification of marker expression. METHODS: We compared three commonly used methods of ENS wholemount dissection: two flat-sheet preparations that differed in the order of microdissection and fixation and a third rod-mounted peeling technique. We also tested a reversed orientation variation of flat-sheet peeling, two step-by-step variations of the rod peeling technique, and whole-gut fixation as a tube. We assessed marker expression using immunohistochemistry, genetic reporter lines, confocal microscopy, and automated image analysis. KEY RESULTS AND CONCLUSIONS: We found no significant differences between the two flat-sheet preparation methods in the expression of calretinin or neuronal nitric oxide synthase (nNOS) as a proportion of total neurons in ileum myenteric plexus. However, the rod-mounted peeling method resulted in decreased proportion of neurons labeled for both calretinin and nNOS. This method also resulted in decreased transgenic reporter fluorescent protein (tdTomato) for substance P in distal colon and choline acetyltransferase (ChAT) in both ileum and distal colon. These results suggest that labeling among some markers, both native protein and transgenic fluorescent reporters, is decreased by the rod-mounted mechanical method of peeling. The step-by-step variations of this method point to mechanical manipulation of the tissue as the likely cause of decreased labeling. Our study thereby demonstrates a critical variability in wholemount muscularis dissection methods.

RET Signaling Persists in the Adult Intestine and Stimulates Motility by Limiting PYY Release From Enteroendocrine Cells.

BACKGROUND & AIMS: RET tyrosine kinase is necessary for enteric nervous system development. Loss-of-function RET mutations cause Hirschsprung disease (HSCR), in which infants are born with aganglionic bowel. Despite surgical correction, patients with HSCR often experience chronic defecatory dysfunction and enterocolitis, suggesting that RET is important after development. To test this hypothesis, we determined the location of postnatal RET and its significance in gastrointestinal (GI) motility. METHODS: RetCFP/+ mice and human transcriptional profiling data were studied to identify the enteric neuronal and epithelial cells that express RET. To determine whether RET regulates gut motility in vivo, genetic, and pharmacologic approaches were used to disrupt RET in all RET-expressing cells, a subset of enteric neurons, or intestinal epithelial cells. RESULTS: Distinct subsets of enteric neurons and enteroendocrine cells expressed RET in the adult intestine. RET disruption in the epithelium, rather than in enteric neurons, slowed GI motility selectively in male mice. RET kinase inhibition phenocopied this effect. Most RET+ epithelial cells were either enterochromaffin cells that release serotonin or L-cells that release peptide YY (PYY) and glucagon-like peptide 1 (GLP-1), both of which can alter motility. RET kinase inhibition exaggerated PYY and GLP-1 release in a nutrient-dependent manner without altering serotonin secretion in mice and human organoids. PYY receptor blockade rescued dysmotility in mice lacking epithelial RET. CONCLUSIONS: RET signaling normally limits nutrient-dependent peptide release from L-cells and this activity is necessary for normal intestinal motility in male mice. These effects could contribute to dysmotility in HSCR, which predominantly affects males, and uncovers a mechanism that could be targeted to treat post-prandial GI dysfunction.