Cells of the human intestinal tract mapped across space and time.

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.

Morphological changes of the myenteric plexus at different gut segments of human fetuses.

The neural crest cell-derived enteric nervous system (ENS) is the intrinsic innervation of the gastrointestinal tract (GIT) which consists of neurons and enteric glia cells in the myenteric ganglia and forming plexus. The ENS consists mainly of submucosal and myenteric plexuses. It has various functions on the GIT, which include control of local blood flow, motility, mucosal transport, secretions, immune modulation as well as endocrine functions and coordinated contractile activity of smooth muscle. The knowledge on the development of the innervations at different segments of the gut in humans from 11 to 26 weeks of gestation (WG) may help in understanding the pathophysiology of various congenital diseases affecting the ENS. The aim of this study is to determine the morphology of the myenteric plexus in the esophagus, ascending colon and sigmoid colon at various weeks of gestation. Tissue samples from 10 naturally terminated fetuses aged 11-26 WG were processed for hematoxylin and eosin staining and immunohistochemistry assay. The neurons, enteric glia, the smooth muscle were visualized using PGP9.5, Vimentin and S-100 antibodies. The number of neurons and enteric glial cells appeared lowest in the esophagus than the ascending and sigmoid colon. The myenteric ganglion was closely apposed to each other, forming a continuous arch along the entire circumference of gut sections of ascending and sigmoid colon but the myenteric ganglia in the esophagus was thinly populated and widely spread in the fetus at 13 WG. As the fetal gastrointestinal tract grew in diameter and length, the myenteric ganglia became discernible.

Quantification of the neurons of myenteric plexus of the bat molossus rufus / Quantificação dos neurônios do plexo mientérico de morcegos da espécie Molossus rufus

There are no studies that characterize the enteric nervous system (ENS) bats. The organization and density of myenteric neurons may vary according to the animal species, as well as the segment of the digestive tube considered. The nitric oxide is one of the key neurotransmitters present in the myenteric neurons, acting as a mediator in the smooth muscle relaxation. These neurons are evidenced by immunohistochemistry of nitric oxide synthase (NOS) or by NADPH-diaphorase histochemistry. In this sense, this study aimed to characterize the total neuronal population and subpopulation NADPH-d+ of the myenteric plexus present in the jejunum of the insectivore species Molossus rufus quantitatively. Five specimens were collected of M. rufus in a buffer area of the "Reserva Biológica das Perobas" in the microregion of Cianorte/PR. After the euthanasia, in a chamber saturated with isoflurane, segments were collected from the small intestine corresponding to the jejunum intended for two techniques for neuronal marking, Giemsa and NADPH-diaphorase, and a fragment to the histological technique of hematoxylin-eosin and Masson's trichrome. All the procedures were approved by the "Comitê de Ética no Uso de Animais Unipar" (CEUA - protocol No. 34347/2017) and the "Instituto Chico Mendes de Conservação da Biodiversidade" (ICMBio - protocol No. 60061-1) The histological sections allowed to highlight the location of the myenteric plexus between the longitudinal and circular layers of the muscular tunic. The myenteric plexus had an average of total neuronal population (neurons Giemsa+) of 279.23 neurons/mm2, being the nitrergic neurons (neurons NADPH-d+) represented 20.4% of this total population, with an average of 58.14 neuron/mm2. Therefore, the collected data are consistent with previous studies in other mammalian species concerning the location of the myenteric plexus, as well as the neural myenteric proportion NADPH-d+ compared with the population of neurons Giemsa+. The gaps in the knowledge of ENS of bats limits comparative intraspecific and interspecific studies.(AU)

Não há estudos que caracterizem o sistema nervoso entérico (SNE) destes animais, configurando uma lacuna no conhecimento quanto à biologia destes indivíduos. A organização e densidade dos neurônios mientéricos podem variar de acordo com a espécie animal bem como o segmento do tubo digestório considerado. O óxido nítrico é um dos principais neurotransmissores presentes nos neurônios mientéricos, atuando como mediador no relaxamento do músculo liso gastrointestinal, de modo que estes neurônios são evidenciados igualmente pela imunohistoquímica da óxido nítrico-sintase (NOS) ou pela histoquímica da NADPH-diaforase. Neste sentido, objetivou-se caracterizar quantitativamente a população neuronal total e subpopulação NADPH-d+ do plexo mientérico presente no jejuno da espécie Molossus rufus de hábito alimentar insetívoro. Foram coletados cinco espécimes de M. rufus em área de amortecimento da Reserva Biológica das Perobas na microrregião de Cianorte/PR. Após a eutanásia, em câmara saturada com isoflurano, foram coletados segmentos do intestino delgado correspondentes ao jejuno destinados a duas técnicas para marcação neuronal, Giemsa e NADPH-diaforase e, um fragmento para a técnica histológica de hematoxilina-eosina e tricômio de Masson. Todos os procedimentos realizados foram aprovados pelo Comitê de Ética no Uso de Animais da Unipar (CEUA - protocolo nº 34347/2017) e pelo Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio - protocolo nº 60061-1) Os cortes histológicos possibilitaram evidenciar a localização do plexo mientérico entre os estratos longitudinal e circular da túnica muscular. Neurônios Giemsa+ apresentaram uma média de 279,23 neurônios/mm2, já os neurônios nitrérgicos apresentaram em média 20,4% da população neuronal mientérica total, sendo evidenciados 58,14 neurônios NADPH-d+/mm2. Portanto, os dados coletados mostram-se condizentes com estudos anteriores em outras espécies de mamíferos quanto à localização do plexo mientérico, bem como, a proporção neuronal mientérica NADPH-d+ comparada com a população de neurônios Giemsa+. As lacunas existentes quanto ao conhecimento do SNE de morcegos limita possíveis inferências em comparativo intraespecífico e interespecífico.(AU)

The Enteric Nervous System for Epithelial Researchers: Basic Anatomy, Techniques, and Interactions With the Epithelium.

The intestinal epithelium does not function in isolation, but interacts with many components including the Enteric Nervous System (ENS). Understanding ENS and intestinal epithelium interactions requires multidisciplinary approaches to uncover cells involved, mechanisms used, and the ultimate influence on intestinal physiology. This review is intended to serve as a reference for epithelial biologists interested in studying these interactions. With this in mind, this review aims to summarize the basic anatomy of the epithelium and ENS, mechanisms by which they interact, and techniques used to study these interactions. We highlight in vitro, ex vivo and in vivo techniques. Additionally, ENS influence on epithelial proliferation and gene expression within stem and differentiated cells as well as gastrointestinal cancer are discussed.

Engineering a second brain in a dish.

The utilization of human pluripotent stem cells holds great promise in elucidating principles of developmental biology and applications in personalized and regenerative medicine. Breakthroughs from the last decade have allowed the scientific community to better understand and successfully manipulate human pluripotent stem cells using distinct differentiation strategies into a variety of target tissues. This manipulation relies solely on our understanding of developmental processes occurring in model organisms. The in vitro translation of our developmental knowledge upon stem cells provides a new means to generate specific tissue to understand developmental and disease mechanisms, as well as physiological processes. The generation of an integrated human intestinal tissue is one such example. In this review, we highlight the biological motivation behind the generation of human intestinal organoids. We further describe the integration of an enteric nervous system within the organoid to generate a functional intestine. Forthcoming strategies to add additional complexities to the intestinal tissue so as to better understand how our "second brain" functions within the gut are also discussed. The organoid system offers a promising avenue to understand how the enteric nervous system works and patterns the human intestine during both physiology and disease.

Morphological evidence for novel enteric neuronal circuitry in guinea pig distal colon.

The gastrointestinal (GI) tract is unique compared to all other internal organs; it is the only organ with its own nervous system and its own population of intrinsic sensory neurons, known as intrinsic primary afferent neurons (IPANs). How these IPANs form neuronal circuits with other functional classes of neurons in the enteric nervous system (ENS) is incompletely understood. We used a combination of light microscopy, immunohistochemistry and confocal microscopy to examine the topographical distribution of specific classes of neurons in the myenteric plexus of guinea-pig colon, including putative IPANs, with other classes of enteric neurons. These findings were based on immunoreactivity to the neuronal markers, calbindin, calretinin and nitric oxide synthase. We then correlated the varicose outputs formed by putative IPANs with subclasses of excitatory interneurons and motor neurons. We revealed that calbindin-immunoreactive varicosities form specialized structures resembling 'baskets' within the majority of myenteric ganglia, which were arranged in clusters around calretinin-immunoreactive neurons. These calbindin baskets directly arose from projections of putative IPANs and represent morphological evidence of preferential input from sensory neurons directly to a select group of calretinin neurons. Our findings uncovered that these neurons are likely to be ascending excitatory interneurons and excitatory motor neurons. Our study reveals for the first time in the colon, a novel enteric neural circuit, whereby calbindin-immunoreactive putative sensory neurons form specialized varicose structures that likely direct synaptic outputs to excitatory interneurons and motor neurons. This circuit likely forms the basis of polarized neuronal pathways underlying motility.

Intestinal smooth muscle is required for patterning the enteric nervous system.

The development of the enteric nervous system (ENS) and intestinal smooth muscle occurs in a spatially and temporally correlated manner, but how they influence each other is unknown. In the developing mid-gut of the chick embryo, we find that α-smooth muscle actin expression, indicating early muscle differentiation, occurs after the arrival of migrating enteric neural crest-derived cells (ENCCs). In contrast, hindgut smooth muscle develops prior to ENCC arrival. Smooth muscle development is normal in experimentally aganglionic hindguts, suggesting that proper development and patterning of the muscle layers does not rely on the ENS. However, inhibiting early smooth muscle development severely disrupts ENS patterning without affecting ENCC proliferation or apoptosis. Our results demonstrate that early intestinal smooth muscle differentiation is required for patterning the developing ENS.

Morphoquantitative study of Rattus norvegicus submucosal plexus by different neuronal evidentiation histochemical techniques / Estudio morfocuantitativo del plexo submucoso de Rattus norvegicus por medio de diferentes técnicas histoquímicas para verificar características neuronales

Enteric nervous plexuses have been the object of several studies, specially the myenteric plexus whose studies describe its organization, functions and alterations. On the other hand, the submucosal plexus has been less studied and still needs descriptive studies. To analyze morphologically and quantitatively submucosal neurons of the jejunum of 90-day-old healthy rats using different techniques for neuronal staining as a way to provide normality data to compare with future experimental studies. Whole mount preparations of the jejunum were submitted to Giemsa, NADH-diaphorase and NADPH-diaphorase techniques to stain the total neuronal population, more metabolically active subpopulation and subpopulation of nitrergic neurons, respectively. Neurons of the submucosal plexus of adult rats are mainly organized in ganglia with varied sized and shapes. Giemsa technique stained 243.93 ± 7.68 neurons per mm2. Regarding the total population stained by Giemsa, NADH- diaphorase positive (139.09 ± 11.14/mm2) neurons represented 57 % and NADPH-diaphorase positive (18.17 ± 0.28/mm2) represented 7.5 %. The area of the cell body was bigger in nitrergic neurons (412.29 ± 150.22) than in the ones stained by Giemsa (254.71 ± 63.32) and NADH-diaphorase positive (243.98 ± 123.82).

El plexo nervioso entérico ha sido objeto de varios estudios, especialmente el plexo mientérico, cuyos estudios consisten en describir su organización, funciones y alteraciones. Por otro lado, el plexo submucoso ha sido menos investigado y todavía necesita estudios descriptivos. Para analizar morfológica y cuantitativamente las neuronas de la submucosa del yeyuno de ratas de 90 días de edad, se realizaron diferentes técnicas de tinción neuronales, en animales sanos, como una forma de proporcionar datos de normalidad y compararlo con futuros estudios experimentales. Se realizaron montajes con preparados enteros del yeyuno que fueron sometidos a las técnicas de Giemsa, de NADPH-diaforasa y NADH-diaforasa para teñir la población total neuronal, subpoblación más activa metabólicamente y subpoblación de neuronas nitrérgicas, respectivamente. Las neuronas del plexo submucoso de ratas adultas se organizan principalmente en los ganglios con variaciones de tamaño y formas. Con la técnica de Giemsa se tiñeron 243.93±7.68 neuronas por mm2. Con respecto a la población total teñida con Giemsa, fueron positivas para NADH- diaforasa en 139.09 ±11.14 / mm2 neuronas, representando el 57% y fueron positivas para NADPH-diaforasa en 18,17 ± 0,28 / mm2 neuronas, lo que representó el 7,5%. El área del cuerpo celular fue mayor en neuronas nitrérgicas (412,29 ± 150.22) que en las teñidas con Giemsa (254,71 ± 63,32) y NADH-diaforasa positivas (243,98 ± 123,82).

Development of the intrinsic and extrinsic innervation of the gut.

The gastrointestinal (GI) tract is innervated by intrinsic enteric neurons and by extrinsic efferent and afferent nerves. The enteric (intrinsic) nervous system (ENS) in most regions of the gut consists of two main ganglionated layers; myenteric and submucosal ganglia, containing numerous types of enteric neurons and glial cells. Axons arising from the ENS and from extrinsic neurons innervate most layers of the gut wall and regulate many gut functions. The majority of ENS cells are derived from vagal neural crest cells (NCCs), which proliferate, colonize the entire gut, and first populate the myenteric region. After gut colonization by vagal NCCs, the extrinsic nerve fibers reach the GI tract, and Schwann cell precursors (SCPs) enter the gut along the extrinsic nerves. Furthermore, a subpopulation of cells in myenteric ganglia undergoes a radial (inward) migration to form the submucosal plexus, and the intrinsic and extrinsic innervation to the mucosal region develops. Here, we focus on recent progress in understanding the developmental processes that occur after the gut is colonized by vagal ENS precursors, and provide an up-to-date overview of molecular mechanisms regulating the development of the intrinsic and extrinsic innervation of the GI tract.

Nontoxicity of lentiviral vector infection to viability, migration, apoptosis, and differentiation of postnatal rat enteric neural crest-derived cells.

Lentiviral vector infection of enhanced green fluorescent protein fluorescence reporter genes in enteric neural crest-derived cells maintained efficient, stable, long-term labeling and the infected enteric neural crest-derived cells could survive, proliferate, and express fluorescent reporter genes. However, the method does not show whether there is some defined or undefined toxicity to the enteric neural crest-derived cells, which may affect enteric neural crest-derived cells' properties. Here, we evaluated the enteric neural crest-derived cells properties under the influence of lentivirus infection of enhanced green fluorescent protein fluorescence reporter genes. This study used the cell count kit-8 for measurement of vitality, transwell for cell migration, immunocytochemistry for cell count and identification, and tested the apoptosis of the enteric neural crest-derived cells with flow cytometry. The enteric neural crest-derived cells with or without lentivirus and their derivative enteric neural crest-derived cells could form characteristic neurospheres, and maintain their level of fluorescent label steady. When cultured under inducing conditions, enteric neural crest-derived cells differentiated into neurons and glia. The results showed that the enteric neural crest-derived cells with or without lentivirus showed no significant difference in viability, migration, apoptosis, neuronal, and glial ratio. The study identified that lentivirus can be used in a nontoxic manner for infection of enhanced green fluorescent protein fluorescence reporter genes into enteric neural crest-derived cells.

Enteric neuroplasticity in seawater-adapted European eel (Anguilla anguilla).

European eels live most of their lives in freshwater until spawning migration to the Sargasso Sea. During seawater adaptation, eels modify their physiology, and their digestive system adapts to the new environment, drinking salt water to compensate for the continuous water loss. In that period, eels stop feeding until spawning. Thus, the eel represents a unique model to understand the adaptive changes of the enteric nervous system (ENS) to modified salinity and starvation. To this purpose, we assessed and compared the enteric neuronal density in the cranial portion of the intestine of freshwater eels (control), lagoon eels captured in brackish water before their migration to the Sargasso Sea (T0), and starved seawater eels hormonally induced to sexual maturity (T18; 18 weeks of starvation and treatment with standardized carp pituitary extract). Furthermore, we analyzed the modification of intestinal neuronal density of hormonally untreated eels during prolonged starvation (10 weeks) in seawater and freshwater. The density of myenteric (MP) and submucosal plexus (SMP) HuC/D-immunoreactive (Hu-IR) neurons was assessed in wholemount preparations and cryosections. The number of MP and SMP HuC/D-IR neurons progressively increased from the freshwater to the salty water habitat (control > T0 > T18; P < 0.05). Compared with freshwater eels, the number of MP and SMP HuC/D-IR neurons significantly increased (P < 0.05) in the intestine of starved untreated salt water eels. In conclusion, high salinity evokes enteric neuroplasticity as indicated by the increasing number of HuC/D-IR MP and SMP neurons, a mechanism likely contributing to maintaining the body homeostasis of this fish in extreme conditions.

Development of myenteric cholinergic neurons in ChAT-Cre;R26R-YFP mice.

Cholinergic neurons are the major excitatory neurons of the enteric nervous system (ENS), and include intrinsic sensory neurons, interneurons, and excitatory motor neurons. Cholinergic neurons have been detected in the embryonic ENS; however, the development of these neurons has been difficult to study as they are difficult to detect prior to birth using conventional immunohistochemistry. In this study we used ChAT-Cre;R26R-YFP mice to examine the development of cholinergic neurons in the gut of embryonic and postnatal mice. Cholinergic (YFP+) neurons were first detected at embryonic day (E)11.5, and the proportion of cholinergic neurons gradually increased during pre- and postnatal development. At birth, myenteric cholinergic neurons comprised less than half of their adult proportions in the small intestine (25% of myenteric neurons were YFP+ at P0 compared to 62% in adults). The earliest cholinergic neurons appear to mainly project anally. Projections into the presumptive circular muscle were first observed at E14.5. A subpopulation of cholinergic neurons coexpress calbindin through embryonic and postnatal development, but only a small proportion coexpressed neuronal nitric oxide synthase. Our study shows that cholinergic neurons in the ENS develop over a protracted period of time.

Nerves in the intersphincteric space of the human anal canal with special reference to their continuation to the enteric nerve plexus of the rectum.

In the intersphincteric space of the anal canal, nerves are thought to "change" from autonomic to somatic at the level of the squamous-columnar epithelial junction of the anal canal. To compare the nerve configuration in the intersphincteric space with the configuration in adjacent areas of the human rectum, we immunohistochemically assessed tissue samples from 12 donated cadavers, using antibodies to S100, neuronal nitric oxide synthase (nNOS), and tyrosine hydroxylase (TH). Antibody to S100 revealed a clear difference in intramuscular nerve distribution patterns between the circular and longitudinal muscle layers of the most inferior part of the rectum, with the former having a plexus-like configuration, while the latter contained short, longitudinally running nerves. Most of the intramural ganglion cells in the anal canal were restricted to above the epithelial junction, but some were located just below that level. Near or at the level of the epithelial junction, the nerves along the rectal adventitia and Auerbach's nerve plexus joined to form intersphincteric nerves, with all these nerves containing both nNOS-positive parasympathetic and TH-positive sympathetic nerve fibers. Thus, it was histologically difficult to distinguish somatic intersphincteric nerves from the autonomic Auerbach's plexus. In the intersphincteric space, the autonomic nerve elements with intrapelvic courses seemed to "borrow" a nerve pathway in the peripheral branches of the pudendal nerve. Injury to the intersphincteric nerve during surgery may result in loss of innervation in the major part of the internal anal sphincter.

3-D imaging and illustration of mouse intestinal neurovascular complex.

Because of the dispersed nature of nerves and blood vessels, standard histology cannot provide a global and associated observation of the enteric nervous system (ENS) and vascular network. We prepared transparent mouse intestine and combined vessel painting and three-dimensional (3-D) neurohistology for joint visualization of the ENS and vasculature. Cardiac perfusion of the fluorescent wheat germ agglutinin (vessel painting) was used to label the ileal blood vessels. The pan-neuronal marker PGP9.5, sympathetic neuronal marker tyrosine hydroxylase (TH), serotonin, and glial markers S100B and GFAP were used as the immunostaining targets of neural tissues. The fluorescently labeled specimens were immersed in the optical clearing solution to improve photon penetration for 3-D confocal microscopy. Notably, we simultaneously revealed the ileal microstructure, vasculature, and innervation with micrometer-level resolution. Four examples are given: 1) the morphology of the TH-labeled sympathetic nerves: sparse in epithelium, perivascular at the submucosa, and intraganglionic at myenteric plexus; 2) distinct patterns of the extrinsic perivascular and intrinsic pericryptic innervation at the submucosal-mucosal interface; 3) different associations of serotonin cells with the mucosal neurovascular elements in the villi and crypts; and 4) the periganglionic capillary network at the myenteric plexus and its contact with glial fibers. Our 3-D imaging approach provides a useful tool to simultaneously reveal the nerves and blood vessels in a space continuum for panoramic illustration and analysis of the neurovascular complex to better understand the intestinal physiology and diseases.

Morphological evidence of local reflex arc in the rat's tongue.

Lingual components of the autonomic nervous system are considered to be the most rostral portion of the enteric nervous system. Therefore our aim was to study the intrinsic nerve cell bodies and synapses using immunohisto-, immunocytochemical methods. Several small groups of ganglia with cell bodies immunoreactive (IR) for vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and substance P (SP) were observed just below the gustatory epithelium. A few somatostatin and galanin IR nerve cell bodies were also found. Many IR cell bodies were also demonstrated in the glands and next to blood vessels. Some of these cell bodies were multipolar and some of them were small neurons with an ovoid shape having only one process. Cell bodies positive for calcitonin gene-related peptide (CGRP) were detected neither in the superficial nor in the deep portion. Electronmicroscopical analysis demonstrated different IR nerve fibres having axo-somatic and axo-dendritic synapses with other immunonegative cells. In a few cases VIP IR nerve processes were found to synaptize with other VIP positive nerve cell bodies. These results support the existance of intralingual reflex in the tongue, where the ganglia might have an integrative role of the different neuropeptide containing nerve fibres.

[Enteric nervous system and Parkinson's disease]. / Système nerveux entérique et maladie de Parkinson.

It has become increasingly evident over the last years that Parkinson's disease is a multicentric neurodegenerative disease that affects several neuronal structures outside the substantia nigra, among which is the enteric nervous system. The aims of the present article are to discuss the role of the enteric nervous system lesions in pathology spreading (Braak's hypothesis) and in the gastrointestinal dysfunction encountered in Parkinson's disease. Owing to its accessibility to biopsies, we further discuss the use of the enteric nervous system as an original source of biomarker in Parkinson's disease.

Nonruminant Nutrition Symposium: Neurogastroenterology and food allergies.

Neurogastroenterology is a subspecialty encompassing relations of the nervous system to the gastrointestinal tract. The central concept is emergence of whole organ behavior from coordinated activity of the musculature, mucosal epithelium, and blood vasculature. Behavior of each effector is determined by the enteric nervous system (ENS). The ENS is a minibrain positioned close to the effectors it controls. The ENS neurophysiology is in the framework of neurogastroenterology. The digestive tract is recognized as the largest lymphoid organ in the body with a unique complement of mast cells. In its position at the "dirtiest" of interfaces between the body and outside world, the mucosal immune system encounters food antigens, bacteria, parasites, viruses, and toxins. Epithelial barriers are insufficient to exclude fully the antigenic load, thereby allowing chronic challenges to the immune system. Observations in antigen-sensitized animals document direct communication between the mucosal immune system and ENS. Communication is functional and results in adaptive responses to circumstances within the lumen that are threatening to the functional integrity of the whole animal. Communication is paracrine and incorporates specialized sensing functions of mast cells for specific antigens together with the capacity of the ENS for intelligent interpretation of the signals. Immuno-neural integration progresses sequentially, beginning with immune detection, followed by signal transfer to the ENS, followed by neural interpretation and then selection of a neural program with coordinated mucosal secretion and a propulsive motor event that quickly clears the threat from the intestinal lumen. Operation of the defense program evokes symptoms of cramping abdominal pain, fecal urgency, and acute watery diarrhea. Investigative approaches to immuno-ENS interactions merge the disciplines of mucosal immunology and ENS neurophysiology into the realm of neurogastroenterology.

Targeted deletion of Hand2 in enteric neural precursor cells affects its functions in neurogenesis, neurotransmitter specification and gangliogenesis, causing functional aganglionosis.

Targeted deletion of the bHLH DNA-binding protein Hand2 in the neural crest, impacts development of the enteric nervous system (ENS), possibly by regulating the transition from neural precursor cell to neuron. We tested this hypothesis by targeting Hand2 deletion in nestin-expressing neural precursor (NEP) cells. The mutant mice showed abnormal ENS development, resulting in lethal neurogenic pseudo-obstruction. Neurogenesis of neurons derived from NEP cells identified a second nestin non-expressing neural precursor (NNEP) cell in the ENS. There was substantial compensation for the loss of neurons derived from the NEP pool by the NNEP pool but this was insufficient to abrogate the negative impact of Hand2 deletion. Hand2-mediated regulation of proliferation affected both neural precursor and neuron numbers. Differentiation of glial cells derived from the NEP cells was significantly decreased with no compensation from the NNEP pool of cells. Our data indicate differential developmental potential of NEPs and NNEPs; NNEPs preferentially differentiate as neurons, whereas NEPs give rise to both neurons and glial cells. Deletion of Hand2 also resulted in complete loss of NOS and VIP and a significant decrease in expression of choline acetyltransferase and calretinin, demonstrating a role for Hand2 in neurotransmitter specification and/or expression. Loss of Hand2 resulted in a marked disruption of the developing neural network, exemplified by lack of a myenteric plexus and extensive overgrowth of fibers. Thus, Hand2 is essential for neurogenesis, neurotransmitter specification and neural network patterning in the developing ENS.

Optical clearing improves the imaging depth and signal-to-noise ratio for digital analysis and three-dimensional projection of the human enteric nervous system.

BACKGROUND: Due to the dispersed nature of neurites and fibers, the microtome-based 2-dimensional histology provides only a limited perspective of the enteric nervous system. To visualize the enteric plexus, we applied optical clearing to avoid scattering in the human ileum to facilitate photon penetration for 3-dimensional (3-D) microscopy of the neural tissue. METHODS: Human ileal specimens were derived by trimming the donor bowel due to its excess length during the clinical trial of small intestinal transplantation. The pan-neuronal marker PGP9.5 was used as the immunostaining target to reveal the enteric plexuses. The labeled tissues were immersed in the optical-clearing solution prior to deep-tissue confocal microscopy. The serial sections were digitally analyzed and processed by reconstruction algorithms for 3-D visualization. KEY RESULTS: Optical clearing of the ileal specimen led to less fluorescence signal decay along the focal path in the tissue and a higher signal-to-noise ratio of the confocal micrographs in comparison with the untreated saline control. Taking advantage of the high signal-to-noise ratio images, we applied software-based signal analysis to identify the presence of the nerve fibers and quantify the signal peaks. The image stacks derived from the serial anatomic micrographs created panoramic views of the gut wall innervations with their associated microstructures. CONCLUSIONS & INFERENCES: We provide an optical approach to improve the imaging depth in 3-D neurohistology of the human ileum. This methodology has significant promise in facilitating our understanding of the enteric nervous system in health and disease.

Structural and functional components of the feline enteric nervous system.

Neurohistological and immunohistochemical examinations of the feline enteric nervous system (ENS) were performed by using antibodies against neuron-specific enolase (NSE), phosphorylated neurofilaments (PN), non-phosphorylated neurofilaments (NPN) and vasoactive intestinal peptide (VIP), whereas glial cells were investigated by using antibodies against glial fibrillary acidic protein (GFAP). The study included full-thickness biopsies of the stomach, duodenum, jejunum, ileum and colon of 11 healthy cats. In this study, immunohistochemical staining of feline ENS with antibodies to NSE, PN and NPN revealed the presence of different ganglionated and aganglionated plexus. The two ganglionated plexus were arranged in a plexus submucosus internus & externus and a plexus myentericus. Furthermore, plexus mucosus and subserosal plexus represented two aganglionated plexus. GFAP-stained cellular elements were smaller than and in close contact to enteric neurons possibly resembling astrocytes of the central nervous system. VIP is one of the major neurotransmitters of enteric inhibitory neurons, and immunoreactivity was present in all layers of the gut, especially in ganglionated plexus. This is the first report, describing feline ENS by using immunohistochemical methods.