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.

Distribution, quantification, and characterization of substance P enteric neurons in the submucosal and myenteric plexuses of the porcine colon.

The pig is an important translational model for studying intestinal physiology and disorders for its many homologies with humans, including the organization of the enteric nervous system (ENS), the major regulator of gastrointestinal functions. This study focused on the quantification and neurochemical characterization of substance P (SP) neurons in the pig ascending (AC) and descending colon (DC) in wholemount preparations of the inner submucosal plexus (ISP), outer submucosal plexus (OSP), and myenteric plexus (MP). We used antibodies for the pan-neuronal marker HuCD, and choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS), markers for excitatory and inhibitory transmitters, for multiple labeling immunofluorescence and high-resolution confocal microscopy. The highest density of SP immunoreactive (IR) neurons was in the ISP (222/mm2 in the AC, 166/mm2 in the DC), where they make up about a third of HuCD-IR neurons, compared to the OSP and MP (19-22% and 13-17%, respectively, P < 0.001-0.0001). HuCD/SP/ChAT-IR neurons (up to 23%) were overall more abundant than HuCD/SP/nNOS-IR neurons (< 10%). Most SP-IR neurons contained ChAT-IR (62-85%), whereas 18-38% contained nNOS-IR with the highest peak in the OSP. A subpopulation of SP-IR neurons contains both ChAT- and nNOS-IR with the highest peak in the OSP and ISP of DC (33-36%) and the lowest in the ISP of AC (< 10%, P < 0.001). SP-IR varicose fibers were abundant in the ganglia. This study shows that SP-IR neurons are functionally distinct with variable proportions in different plexuses in the AC and DC reflecting diverse functions of specific colonic regions.

Annotated translation of Georg Meissner's first description of the submucosal plexus.

BACKGROUND: The discovery and detailed descriptions of the enteric nervous system dates back only ˂200 years. The 19th century was a golden age of histological, morphological, and physiological breakthroughs propelled by technological advances in microscopy, electricity, and scientific methodology. As a matter of fact, German-speaking scientists were highly successful during this period as can still be appreciated by the sheer number of German eponyms in anatomy. Therefore, the main language in scientific literature of this field was German at the time, thus, limiting the accessibility to the publications and scientific discussions from back then for the broader English-speaking scientific community today. PURPOSE: Here, an annotated translation of Meissner's first description of the submucosal plexus is provided along with a short biography of Georg Meissner and a review of the scientific literature and controversy surrounding his discovery.

5xFAD mice do not have myenteric amyloidosis, dysregulation of neuromuscular transmission or gastrointestinal dysmotility.

BACKGROUND: Alterations in gastrointestinal (GI) function and the gut-brain axis are associated with progression and pathology of Alzheimer's Disease (AD). Studies in AD animal models show that changes in the gut microbiome and inflammatory markers can contribute to AD development in the central nervous system (CNS). Amyloid-beta (Aß) accumulation is a major AD pathology causing synaptic dysfunction and neuronal death. Current knowledge of the pathophysiology of AD in enteric neurons is limited, and whether Aß accumulation directly disrupts enteric neuron function is unknown. METHODS: In 6-month-old 5xFAD (transgenic AD) and wildtype (WT) male and female mice, GI function was assessed by colonic transit in vivo; propulsive motility and GI smooth muscle contractions ex vivo; electrochemical detection of enteric nitric oxide release in vitro, and changes in myenteric neuromuscular transmission using smooth muscle intracellular recordings. Expression of Aß in the brain and colonic myenteric plexus in these mice was determined by immunohistochemistry staining and ELISA assay. KEY RESULTS: At 6 months, 5xFAD mice did not show significant changes in GI motility or synaptic neurotransmission in the small intestine or colon. 5xFAD mice, but not WT mice, showed abundant Aß accumulation in the brain. Aß accumulation was undetectable in the colonic myenteric plexus of 5xFAD mice. CONCLUSIONS: 5xFAD AD mice are not a robust model to study amyloidosis in the gut as these mice do not mimic myenteric neuronal dysfunction in AD patients with GI dysmotility. An AD animal model with enteric amyloidosis is required for further study.

How big is the little brain in the gut? Neuronal numbers in the enteric nervous system of mice, Guinea pig, and human.

BACKGROUND: Despite numerous studies on the enteric nervous system (ENS), we lack fundamental knowledge on neuronal densities or total neuron numbers in different species. There are more anecdotal than actual figures on nerve counts. METHODS: We used standardized preparation techniques and immunohistochemistry with validated panneuronal markers (human or mouse anti-HuD/C) to determine neuronal densities in specimen from the entire gastrointestinal tract of mice, guinea pig, and humans. In parallel, we measured the dimensions of the gastrointestinal regions in mouse and guinea pig. For humans, we had to rely on literature data. KEY RESULTS: The average neuronal densities along the gastrointestinal tract were 35,011 ± 25,017 1/cm2 for the myenteric and 16,685 ± 9098 1/cm2 for the submucous plexus in mice, 24,315 ± 16,627 and 11,850 ± 6122 1/cm2 for guinea pig myenteric and submucous plexus, respectively, and 21,698 ± 9492 and 16,367 ± 5655 1/cm2 for human myenteric and submucous plexus, respectively. The total number of neurons in the ENS was 2.6 million for mice, 14.6 million for guinea pig, and 168 million for human. CONCLUSIONS & INFERENCES: This study reports the first comprehensive nerve cell count in mice, guinea pig, and human ENS. Neuronal densities were comparable between the three species and the differences in the total numbers of enteric neurons are likely due to body size and intestinal length. The number of enteric neurons is comparable to the number of neurons in the spinal cord for all three species.

Metabotropic 5-HT receptor-mediated effects in the human submucous plexus.

BACKGROUND: Serotonin (5-HT) is an important mediator in the gastrointestinal tract, acting on different neuronal 5-HT receptors. The ionotropic 5-HT3 receptor mediates immediate but transient spike discharge in human enteric neurons. We studied the role of the metabotropic 5-HT1P , 5-HT4 , and 5-HT7 receptors to activate human submucous neurons. METHODS: Neuroimaging using the voltage sensitive dye Di-8-ANEPPS was performed in submucous plexus preparations from human surgical specimens of the small and large intestine. We synthesized a new, stable 5-HT1P agonist, 5-benzyloxyhydrazonoindalpine (5-BOHIP). KEY RESULTS: 5-HT evoked a fast and late-onset spike discharge in enteric neurons. The fast component was blocked by the 5-HT3 receptor antagonist cilansetron, while the remaining sustained response was significantly reduced by the 5-HT1P receptor antagonist 5-hydroxytryptophanyl-5-hydroxytryptophan amide (5-HTP-DP). The newly synthesized 5-HT1P agonist 5-BOHIP induced a slowly developing, long-lasting activation of submucous neurons, which was blocked by 5-HTP-DP. We could not demonstrate any 5-HT7 receptor-induced spike discharge based on the lack of response to 5-carboxamidotryptamine. Similarly, the 5-HT4 agonists 5-methoxytryptamine and prucalopride evoked no immediate or late-onset spike discharge. CONCLUSIONS & INFERENCES: Our work demonstrated for the first time the presence of functional 5-HT1P receptors on human submucous neurons. Furthermore, we found no evidence for a role of 5-HT4 or 5-HT7 receptors in the postsynaptic activation of human submucous neurons by 5-HT.

Isolation of myenteric and submucosal plexus from mouse gastrointestinal tract and subsequent flow cytometry and immunofluorescence.

The myenteric plexus is located between the longitudinal and circular layers of muscularis externa in the gastrointestinal tract. It contains a large network of enteric neurons that form the enteric nervous system (ENS) and control intestinal functions, such as motility and nutrient sensing. This protocol describes the method for physical separation (peeling) of muscularis and submucosal layers of the mouse intestine. Subsequently, the intestinal layers are then processed for flow cytometry and/or immunofluorescence analysis. For complete details on the use and execution of this profile, please refer to Ahrends et al. (2021).

Morphologies, dimensions and targets of gastric nitric oxide synthase neurons.

We investigated the distributions and targets of nitrergic neurons in the rat stomach, using neuronal nitric oxide synthase (NOS) immunohistochemistry and nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry. Nitrergic neurons comprised similar proportions of myenteric neurons, about 30%, in all gastric regions. Small numbers of nitrergic neurons occurred in submucosal ganglia. In total, there were ~ 125,000 neuronal nitric oxide synthase (nNOS) neurons in the stomach. The myenteric cell bodies had single axons, type I morphology and a wide range of sizes. Five targets were identified, the longitudinal, circular and oblique layers of the external muscle, the muscularis mucosae and arteries within the gastric wall. The circular and oblique muscle layers had nitrergic fibres throughout their thickness, while the longitudinal muscle was innervated at its inner surface by fibres of the tertiary plexus, a component of the myenteric plexus. There was a very dense innervation of the pyloric sphincter, adjacent to the duodenum. The muscle strands that run between mucosal glands rarely had closely associated nNOS nerve fibres. Both nNOS immunohistochemistry and NADPH histochemistry showed that nitrergic terminals did not provide baskets of terminals around myenteric neurons. Thus, the nitrergic neuron populations in the stomach supply the muscle layers and intramural arteries, but, unlike in the intestine, gastric interneurons do not express nNOS. The large numbers of nNOS neurons and the density of innervation of the circular muscle and pyloric sphincter suggest that there is a finely graded control of motor function in the stomach by the recruitment of different numbers of inhibitory motor neurons.

Fast synaptic excitatory neurotransmission in the human submucosal plexus.

BACKGROUND: Acetylcholine is the main excitatory neurotransmitter in the enteric nervous system (ENS) in all animal models examined so far. However, data for the human ENS is scarce. METHODS: We used neuroimaging using voltage and calcium dyes, Ussing chamber, and immunohistochemistry to study fast synaptic neurotransmission in submucosal plexus neurons of the human gut. KEY RESULTS: Electrical stimulation of intraganglionic fiber tracts led to fast excitatory postsynaptic potentials (fEPSPs) in 29 submucosal neurons which were all blocked by the nicotinic antagonist hexamethonium. The nicotinic agonist DMPP mimicked the effects of electrical stimulation and had excitatory effects on 56 of 73 neurons. The unselective NMDA antagonist MK-801 blocked fEPSPs in 14 out of 22 neurons as well as nicotine evoked spike discharge. In contrast, the application of NMDA showed only weak effects on excitability or calcium transients. This agreed with the finding that the specific NMDA antagonist D-APV reduced fEPSPs in only 1 out of 40 neurons. Application of AMPA or kainite had no effect in 41 neurons or evoked spike discharge in only one out of 41 neurons, respectively. Immunohistochemistry showed that 98.7 ± 2.4% of all submucosal neurons (n = 6 preparations, 1003 neurons) stained positive for the nicotinic receptor (α1 , α2 or α3 -subunit). Hexamethonium (200 µM) reduced nerve-evoked chloride secretion by 34.3 ± 18.6% (n = 14 patients), whereas D-APV had no effect. CONCLUSION & INFERENCE: Acetylcholine is the most important mediator of fast excitatory postsynaptic transmission in human submucous plexus neurons whereas glutamatergic fEPSPs were rarely encountered.

Small intestine remodeling in male Goto-Kakizaki rats.

BACKGROUND: Obesity is associated with the development of insulin resistance (IR) and type-2 diabetes mellitus (T2DM); however, not all patients with T2DM are obese. The Goto-Kakizaki (GK) rat is an experimental model of spontaneous and non-obese T2DM. There is evidence that the intestine contributes to IR development in GK animals. This information prompted us to investigate small intestine remodeling in this animal model. METHODS: Four-month-old male Wistar (control) and GK rats were utilized for the present study. After removing the small intestine, the duodenum, proximal jejunum, and distal ileum were separated. We then measured villi and muscular and mucosa layer histomorphometry, goblet cells abundance, total myenteric and submucosal neuron populations, and inflammatory marker expression in the small intestinal segments and intestinal transit of both groups of animals. KEY RESULTS: We found that the GK rats exhibited decreased intestinal area (p < 0.0001), decreased crypt depth in the duodenum (p = 0.01) and ileum (p < 0.0001), increased crypt depth in the jejunum (p < 0.0001), longer villi in the jejunum and ileum (p < 0.0001), thicker villi in the duodenum (p < 0.01) and ileum (p < 0.0001), thicker muscular layers in the duodenum, jejunum, and ileum (p < 0.0001), increased IL-1ß concentrations in the duodenum and jejunum (p < 0.05), and increased concentrations of NF-κB p65 in the duodenum (p < 0.01), jejunum and ileum (p < 0.05). We observed high IL-1ß reactivity in the muscle layer, myenteric neurons, and glial cells of the experimental group. GK rats also exhibited a significant reduction in submucosal neuron density in the jejunum and ileum, ganglionic hypertrophy in all intestinal segments studied (p < 0.0001), and a slower intestinal transit (about 25%) compared to controls. CONCLUSIONS: The development of IR and T2DM in GK rats is associated with small intestine remodeling that includes marked alterations in small intestine morphology, local inflammation, and reduced intestinal transit.

Niche-specific functional heterogeneity of intestinal resident macrophages.

Intestinal resident macrophages are at the front line of host defence at the mucosal barrier within the gastrointestinal tract and have long been known to play a crucial role in the response to food antigens and bacteria that are able to penetrate the mucosal barrier. However, recent advances in single-cell RNA sequencing technology have revealed that resident macrophages throughout the gut are functionally specialised to carry out specific roles in the niche they occupy, leading to an unprecedented understanding of the heterogeneity and potential biological functions of these cells. This review aims to integrate these novel findings with long-standing knowledge, to provide an updated overview on our understanding of macrophage function in the gastrointestinal tract and to speculate on the role of specialised subsets in the context of homoeostasis and disease.

Multicolor sparse viral labeling and 3D digital tracing of enteric plexus in mouse proximal colon using a novel adeno-associated virus capsid.

BACKGROUND: Intravenous administration of adeno-associated virus (AAV) can be used as a noninvasive approach to trace neuronal morphology and links. AAV-PHP.S is a variant of AAV9 that effectively transduces the peripheral nervous system. The objective was to label randomly and sparsely enteric plexus in the mouse colon using AAV-PHP.S with a tunable two-component multicolor vector system and digitally trace individual neurons and nerve fibers within microcircuits in three dimensions (3D). METHODS: A vector system including a tetracycline inducer with a tet-responsive element driving three separate fluorophores was packaged in the AAV-PHP.S capsid. The vectors were injected retro-orbitally in mice, and the colon was harvested 3 weeks after. Confocal microscopic images of enteric plexus were digitally segmented and traced in 3D using Neurolucida 360, neuTube, or Imaris software. KEY RESULTS: The transduction of multicolor AAV vectors induced random sparse spectral labeling of soma and neurites primarily in the myenteric plexus of the proximal colon, while neurons in the submucosal plexus were occasionally transduced. Digital tracing in 3D showed various types of wiring, including multiple conjunctions of one neuron with other neurons, neurites en route, and endings; clusters of neurons in close apposition between each other; axon-axon parallel conjunctions; and intraganglionic nerve endings consisting of multiple nerve endings and passing fibers. Most of digitally traced neuronal somas were of small or medium in size. CONCLUSIONS & INFERENCES: The multicolor AAV-PHP.S-packaged vectors enabled random sparse spectral labeling and revealed complexities of enteric microcircuit in the mouse proximal colon. The techniques can facilitate digital modeling of enteric micro-circuitry.

Enteric nervous system and inflammatory bowel diseases: Correlated impacts and therapeutic approaches through the P2X7 receptor.

The enteric nervous system (ENS) consists of thousands of small ganglia arranged in the submucosal and myenteric plexuses, which can be negatively affected by Crohn's disease and ulcerative colitis - inflammatory bowel diseases (IBDs). IBDs are complex and multifactorial disorders characterized by chronic and recurrent inflammation of the intestine, and the symptoms of IBDs may include abdominal pain, diarrhea, rectal bleeding, and weight loss. The P2X7 receptor has become a promising therapeutic target for IBDs, especially owing to its wide expression and, in the case of other purinergic receptors, in both human and model animal enteric cells. However, little is known about the actual involvement between the activation of the P2X7 receptor and the cascade of subsequent events and how all these activities associated with chemical signals interfere with the functionality of the affected or treated intestine. In this review, an integrated view is provided, correlating the structural organization of the ENS and the effects of IBDs, focusing on cellular constituents and how therapeutic approaches through the P2X7 receptor can assist in both protection from damage and tissue preservation.

Role of PD-L1 Expression during the Progression of Submucosal Gastric Cancer.

INTRODUCTION: Programmed death-ligand 1 (PD-L1) expression is a prognostic marker for gastric cancer that correlates with tumor diameter and depth of penetration. But the role of PD-L1 and mechanism(s) employed in the initial phase of invasion in early gastric cancer is yet to be understood. OBJECTIVE: This study aims to elucidate the role of PD-L1 during the progression of gastric cancer, specifically invading the submucosa beyond the lamina muscularis mucosa. METHODS: Using 107 patients with pathological submucosal gastric cancer, we determined the expression of PD-L1 based on the staining of the cell membrane or cytoplasm of tumor cells in the central and invasive front of the tumor. Samples were categorized into 3 groups based on the intensity of PD-L1 expression. CD8+ lymphocytes expressing PD-1 and CD163+ macrophages were used to determine the number of cell nuclei at the invasive front, similar to PD-L1. CMTM6 levels were determined and used to stratify samples into 3 groups. RESULTS: PD-L1 expression was higher in the invasive front (26.2%) than in the central portion of the tumors (7.4%; p < 0.001). Moreover, lymphatic and vascular invasion were more frequently observed in samples with high levels of PD-L1 (lymphatic invasion: 60.7 vs. 35.4%, p = 0.0026, and vascular invasion: 39.3 vs. 16.5%, p = 0.0018). There was no correlation between PD-L1 expression and the levels of PD-1, CD8, CD163, and CMTM6. CONCLUSIONS: PD-L1-expressing cancer cells at the invasive front of gastric cancer influence the initial stages of tumor invasion and lymphovascular permeation in early-stage gastric cancers. Immune checkpoint signaling may be the driving force in the invasive front during the invasion of the submucosa beyond the lamina muscularis mucosa.

Quantitative analysis of enteric neurons containing choline acetyltransferase and nitric oxide synthase immunoreactivities in the submucosal and myenteric plexuses of the porcine colon.

The enteric nervous system (ENS) controls gastrointestinal functions. In large mammals' intestine, it comprises an inner (ISP) and outer (OSP) submucous plexus and a myenteric plexus (MP). This study quantifies enteric neurons in the ISP, OSP, and MP of the pig ascending (AC) and descending colon (DC) using the HuC/D, choline acetyltransferase (ChAT), and neuronal nitric oxide synthase (nNOS) neuronal markers in whole mount preparations with multiple labeling immunofluorescence. We established that the ISP contains the highest number of HuC/D neurons/mm2, which were more abundant in AC vs. DC, followed by OSP and MP with similar density in AC and DC. In the ISP, the density of ChAT immunoreactive (IR) neurons was very similar in AC and DC (31% and 35%), nNOS-IR neurons were less abundant in AC than DC (15% vs. 42%, P < 0.001), and ChAT/nNOS-IR neurons were 5% and 10%, respectively. In the OSP, 39-44% of neurons were ChAT-IR in AC and DC, while 45% and 38% were nNOS-IR and 10-12% were ChAT/nNOS-IR (AC vs. DC P < 0.05). In the MP, ChAT-IR neurons were 44% in AC and 54% in DC (P < 0.05), nNOS-IR neurons were 50% in both, and ChAT/nNOS-IR neurons were 12 and 18%, respectively. The ENS architecture with multilayered submucosal plexuses and the distribution of functionally distinct groups of neurons in the pig colon are similar to humans, supporting the suitability of the pig as a model and providing the platform for investigating the mechanisms underlying human colonic diseases.

Segmental Hypoganglionosis of the Colon: A Case Report.

A 64-year-old woman was admitted to hospital with persistent abdominal pain. She had been hospitalized with similar symptoms on five occasions during a period of 2 years. Computed tomography revealed dilatation and fecal impaction from the ileum to the transverse colon. A barium enema and simultaneous ileus tube radiography showed a narrow segment of descending-sigmoid colon. Colonoscopy showed no mucosal change. Her symptoms did not improve with conservative therapy, so descending and sigmoid colectomy was performed. Histologic examination showed disappearance of ganglion cells; axon of Meissner's plexuses was present, and the number of Auerbach's plexuses was decreased. The definitive diagnosis was segmental hypoganglionosis (SH) of the colon. The postoperative course was uneventful, and the functional result was positive at 1 year postoperatively. SH is extremely rare; however, surgical intervention is expected to be of benefit. Therefore, it is important to keep SH in mind when treating patients with chronic obstruction of the left side of the colon.

The Endocrine Disruptor Bisphenol A (BPA) Affects the Enteric Neurons Immunoreactive to Neuregulin 1 (NRG1) in the Enteric Nervous System of the Porcine Large Intestine.

The enteric nervous system (ENS), located in the wall of the gastrointestinal (GI) tract, is characterized by complex organization and a high degree of neurochemical diversity of neurons. One of the less known active neuronal substances found in the enteric neurons is neuregulin 1 (NRG1), a factor known to be involved in the assurance of normal development of the nervous system. During the study, made up using the double immunofluorescence technique, the presence of NRG1 in the ENS of the selected segment of porcine large intestine (caecum, ascending and descending colon) was observed in physiological conditions, as well as under the impact of low and high doses of bisphenol A (BPA) which is commonly used in the production of plastics. In control animals in all types of the enteric plexuses, the percentage of NRG1-positive neurons oscillated around 20% of all neurons. The administration of BPA caused an increase in the number of NRG1-positive neurons in all types of the enteric plexuses and in all segments of the large intestine studied. The most visible changes were noted in the inner submucous plexus of the ascending colon, where in animals treated with high doses of BPA, the percentage of NRG1-positive neurons amounted to above 45% of all neuronal cells. The mechanisms of observed changes are not entirely clear, but probably result from neurotoxic, neurodegenerative and/or proinflammatory activity of BPA and are protective and adaptive in nature.

Continuum Based Bioelectrical Simulations using Structurally Realistic Gastrointestinal Pacemaker Cell Networks.

Cellular and tissue level bioelectrical activity was simulated over structurally realistic 3D interstitial cell of Cajal (ICC) networks reconstructed from confocal images of a wild type (WT) mouse model with a normal ICC distribution and a Spry4 knockout (KO) mouse model with a mild ICC hyperplasia. First, the ICC pixels within the confocal images were segmented. Then, the segmented images were visually inspected and the 3D surface mesh of the ICC tissue network was created from the 90 slices spanning the myenteric plexus ICC network. After two additional concentric meshes (representing the non-ICC and tissue bath regions) surrounding the ICC region were added, a 3D tetrahedral volume mesh containing the three regions was reconstructed. The electrical propagation through the tissue network was simulated using the bidomain continuum model. The results showed that the ICC network of the WT mouse had a smaller volume than the KO mouse (0.008 vs 0.012 mm3). The simulated bioelectrical activity for both mice showed an isotropic propagation from the initial activation region. Mean velocities of 4.2±1.5 and 4.1±1.3 mm/s were reported for the WT and KO mice, respectively. The velocity in the x-direction was higher than the y-direction for the WT mouse with a percent difference of 14.8%. On the other hand, the velocity in the y-direction was higher for the KO mouse with a percent difference of 9.5%. For both cases, there was no propagation in the z-direction as all the solution points along the same z-depth were simultaneously activated.

Submucosal enteric neurons of the cavine distal colon are sensitive to hypoosmolar stimuli.

KEY POINTS: Neurons of the enteric submucous plexus are challenged by osmolar fluctuations during digestion and absorption of nutrients. Central neurons are very sensitive to changes in osmolality but knowledge on that issue related to enteric neurons is sparse. The present study focuses on investigation of osmosensitivity of submucosal neurons including potential molecular mediating mechanisms. Results show that submucosal neurons respond to hypoosmolar stimuli with increased activity which is partially mediated by the transient receptor potential vanilloid 4 channel. We provided important information on osmosensitive properties of enteric neurons. These data are fundamental to better explain the nerve-mediated control of the gastrointestinal functions during physiological and pathophysiological (diarrhoea) conditions. ABSTRACT: Enteric neurons are located inside the gut wall, where they are confronted with changes in osmolality during (inter-) digestive periods. In particular, neurons of the submucous plexus (SMP), located between epithelial cells and blood vessels may sense and respond to osmotic shifts. The present study was conducted to investigate osmosensitivity of enteric submucosal neurons and the potential role of the transient receptor potential vanilloid 4 channel (TRPV4) as a mediator of enteric neuronal osmosensitivity. Therefore, freshly dissected submucosal preparations from guinea pig colon were investigated for osmosensitivity using voltage-sensitive dye and Ca2+ imaging. Acute hypoosmolar stimuli (final osmolality reached at ganglia of 94, 144 and 194 mOsm kg-1 ) were applied to single ganglia using a local perfusion system. Expression of TRPV4 in the SMP was quantified using qRT-PCR, and GSK1016790A and HC-067047 were used to activate or block the receptor, respectively, revealing its relevance in enteric osmosensitivity. On average, 11.0 [7.0/17.0] % of submucosal neurons per ganglion responded to the hypoosmolar stimulus. The Ca2+ imaging experiments showed that glia responded to the hypoosmolar stimulus, but with a delay in comparison with neurons. mRNA expression of TRPV4 could be shown in the SMP and blockade of the receptor by HC-067047 significantly decreased the number of responding neurons (0.0 [0.0/6.3] %) while the TRPV4 agonist GSK1016790A caused action potential discharge in a subpopulation of osmosensitive enteric neurons. The results of the present study provide insight into the osmosensitivity of submucosal enteric neurons and strongly indicate the involvement of TRPV4 as an osmotransducer.

Human skin-derived precursor cells xenografted in aganglionic bowel.

PURPOSE: One in 5000 newborns is diagnosed with Hirschsprung disease each year in the United States. The potential of employing neural crest stem cells to restore the enteric nervous system has been investigated. Skin-derived precursor cells (SKPs) are multipotent progenitor cells that can differentiate into neurons and gliocytes in vitro and generate enteric ganglion-like structures in rodents. Here we examined the behavior of human SKPs (hSKPs) after their transplantation into a large animal model of colonic aganglionosis. METHODS: Juvenile minipigs underwent a chemical denervation of the colon to establish an aganglionosis model. The hSKPs were generated from human foreskin and were cultured in neuroglial-selective medium. Cells were labeled with a fluorescent dye and were injected into the porcine aganglionic colon. After one week, transplanted hSKPs were assessed by immunofluorescence for markers of multipotency and neuroglial differentiation. RESULTS: In culture, hSKPs expressed nestin and S100b indicative of neuroglial precursors. After xenografting in pigs, hSKPs were identified in the myenteric and submucosal plexuses of the colons. The hSKPs expressed nestin and early neuroglial differentiation markers. CONCLUSIONS: Human SKPs transplanted into aganglionic colon demonstrated immunophenotypes of neuroglial progenitors, suggesting their potential use for Hirschsprung disease.