From diversity to disease: unravelling the role of enteric glial cells.

Enteric glial cells (EGCs) are an essential component of the enteric nervous system (ENS) and play key roles in gastrointestinal development, homeostasis, and disease. Derived from neural crest cells, EGCs undergo complex differentiation processes regulated by various signalling pathways. Being among the most dynamic cells of the digestive system, EGCs react to cues in their surrounding microenvironment and communicate with various cell types and systems within the gut. Morphological studies and recent single cell RNA sequencing studies have unveiled heterogeneity among EGC populations with implications for regional functions and roles in diseases. In gastrointestinal disorders, including inflammatory bowel disease (IBD), infections and cancer, EGCs modulate neuroplasticity, immune responses and tumorigenesis. Recent evidence suggests that EGCs respond plastically to the microenvironmental cues, adapting their phenotype and functions in disease states and taking on a crucial role. They exhibit molecular abnormalities and alter communication with other intestinal cell types, underscoring their therapeutic potential as targets. This review delves into the multifaceted roles of EGCs, particularly emphasizing their interactions with various cell types in the gut and their significant contributions to gastrointestinal disorders. Understanding the complex roles of EGCs in gastrointestinal physiology and pathology will be crucial for the development of novel therapeutic strategies for gastrointestinal disorders.

A call for a unified and multimodal definition of cellular identity in the enteric nervous system.

The enteric nervous system (ENS) is a tantalizing frontier in neuroscience. With the recent emergence of single cell transcriptomic technologies, this rare and poorly understood tissue has begun to be better characterized in recent years. A precise functional mapping of enteric neuron diversity is critical for understanding ENS biology and enteric neuropathies. Nonetheless, this pursuit has faced considerable technical challenges. By leveraging different methods to compare available primary mouse and human ENS datasets, we underscore the urgent need for careful identity annotation, achieved through the harmonization and advancements of wet lab and computational techniques. We took different approaches including differential gene expression, module scoring, co-expression and correlation analysis, unbiased biological function hierarchical clustering, data integration and label transfer to compare and contrast functional annotations of several independently reported ENS datasets. These analyses highlight substantial discrepancies stemming from an overreliance on transcriptomics data without adequate validation in tissues. To achieve a comprehensive understanding of enteric neuron identity and their functional context, it is imperative to expand tissue sources and incorporate innovative technologies such as multiplexed imaging, electrophysiology, spatial transcriptomics, as well as comprehensive profiling of epigenome, proteome, and metabolome. Harnessing human pluripotent stem cell (hPSC) models provides unique opportunities for delineating lineage trees of the human ENS, and offers unparalleled advantages, including their scalability and compatibility with genetic manipulation and unbiased screens. We encourage a paradigm shift in our comprehension of cellular complexity and function in the ENS by calling for large-scale collaborative efforts and research investments.

Organelle mapping in dendrites of human iPSC-derived neurons reveals dynamic functional dendritic Golgi structures.

Secretory pathways within dendrites of neurons have been proposed for local transport of newly synthesized proteins. However, little is known about the dynamics of the local secretory system and whether the organelles are transient or stable structures. Here, we quantify the spatial and dynamic behavior of dendritic Golgi and endosomes during differentiation of human neurons generated from induced pluripotent stem cells (iPSCs). In early neuronal development, before and during migration, the entire Golgi apparatus transiently translocates from the soma into dendrites. In mature neurons, dynamic Golgi elements, containing cis and trans cisternae, are transported from the soma along dendrites, in an actin-dependent process. Dendritic Golgi outposts are dynamic and display bidirectional movement. Similar structures were observed in cerebral organoids. Using the retention using selective hooks (RUSH) system, Golgi resident proteins are transported efficiently into Golgi outposts from the endoplasmic reticulum. This study reveals dynamic, functional Golgi structures in dendrites and a spatial map for investigating dendrite trafficking in human neurons.

Group I Metabotropic Glutamate Receptors Modulate Motility and Enteric Neural Activity in the Mouse Colon.

Glutamate is the major excitatory neurotransmitter in the central nervous system, and there is evidence that Group-I metabotropic glutamate receptors (mGlu1 and mGlu5) have established roles in excitatory neurotransmission and synaptic plasticity. While glutamate is abundantly present in the gut, it plays a smaller role in neurotransmission in the enteric nervous system. In this study, we examined the roles of Group-I mGlu receptors in gastrointestinal function. We investigated the expression of Grm1 (mGlu1) and Grm5 (mGlu5) in the mouse myenteric plexus using RNAscope in situ hybridization. Live calcium imaging and motility analysis were performed on ex vivo preparations of the mouse colon. mGlu5 was found to play a role in excitatory enteric neurotransmission, as electrically-evoked calcium transients were sensitive to the mGlu5 antagonist MPEP. However, inhibition of mGlu5 activity did not affect colonic motor complexes (CMCs). Instead, inhibition of mGlu1 using BAY 36-7620 reduced CMC frequency but did not affect enteric neurotransmission. These data highlight complex roles for Group-I mGlu receptors in myenteric neuron activity and colonic function.

Circadian Control of Gastrointestinal Motility.

With the earth's 24-h rotation cycle, physiological function fluctuates in both diurnal and nocturnal animals, thereby ensuring optimal functioning of the body. The main regulator of circadian rhythm is the suprachiasmatic nucleus (SCN), which is considered the main pacemaker or "central clock" of the body. Located in the anterior hypothalamus, the SCN influences the activity of other brain regions, as well as peripheral organs, through the release of melatonin and corticosteroids. The SCN can be entrained by several cues, with light being the major cue. Light information from the retina is received by the SCN via the retinohypothalamic tract. Non-photic cues such as temperature and exercise can also entrain the SCN, while feeding time can entrain the "molecular clock" contained within peripheral tissues. This enables organs such as the gastrointestinal (GI) tract to coordinate function with environmental factors, such as food availability.The GI tract, which has the main functions of receiving and digesting food, and expelling waste, also shows oscillations in its activity during the circadian cycle. While these changes are evident under normal conditions, GI function is affected when normal circadian rhythm is disrupted. Recent reviews have assessed interactions between the central clock and gut clock; as such, this review aims to focus on the presence of endogenous circadian rhythms in the GI tract, with particular focus to changes to gastrointestinal motility.

Upper Gastrointestinal Motility, Disease and Potential of Stem Cell Therapy.

Many gastrointestinal motility disorders arise due to defects in the enteric nervous system. Achalasia and gastroparesis are two extremely debilitating digestive diseases of the upper gastrointestinal tract caused in part by damage or loss of the nitrergic neurons in the esophagus and stomach. Most current pharmacological and surgical interventions provide no long-term relief from symptoms, and none address the cause. Stem cell therapy, to replace the missing neurons and restore normal gut motility, is an attractive alternative therapy. However, there are a number of hurdles that must be overcome to bring this exciting research from the bench to the bedside.

Enteric glial cells favor accumulation of anti-inflammatory macrophages during the resolution of muscularis inflammation.

Monocyte-derived macrophages (Mφs) are crucial regulators during muscularis inflammation. However, it is unclear which micro-environmental factors are responsible for monocyte recruitment and anti-inflammatory Mφ differentiation in this paradigm. Here, we investigate Mφ heterogeneity at different stages of muscularis inflammation and determine how environmental cues can attract and activate tissue-protective Mφs. Results showed that muscularis inflammation induced marked alterations in mononuclear phagocyte populations associated with a rapid infiltration of Ly6c+ monocytes that locally acquired unique transcriptional states. Trajectory inference analysis revealed two main pro-resolving Mφ subpopulations during the resolution of muscularis inflammation, i.e. Cd206+ MhcIIhi and Timp2+ MhcIIlo Mφs. Interestingly, we found that damage to the micro-environment upon muscularis inflammation resulted in EGC activation, which in turn stimulated monocyte infiltration and the consequent differentiation in anti-inflammatory CD206+ Mφs via CCL2 and CSF1, respectively. In addition, CSF1-CSF1R signaling was shown to be essential for the differentiation of monocytes into CD206+ Mφs and EGC proliferation during muscularis inflammation. Our study provides a comprehensive insight into pro-resolving Mφ differentiation and their regulators during muscularis inflammation. We deepened our understanding in the interaction between EGCs and Mφs, thereby highlighting pro-resolving Mφ differentiation as a potential novel therapeutic strategy for the treatment of intestinal inflammation.

Simultaneous whole-cell patch-clamp and calcium imaging on myenteric neurons.

Live calcium imaging is often used as a proxy for electrophysiological measurements and has been a valuable tool that allows simultaneous analysis of neuronal activity in multiple cells at the population level. In the enteric nervous system, there are two main electrophysiological classes of neurons, after-hyperpolarizing (AH)- and synaptic (S)-neurons, which have been shown to have different calcium handling mechanisms. However, they are rarely considered separately in calcium imaging experiments. A handful of studies have shown that in guinea pig, a calcium transient will accompany a single action potential in AH-neurons, but multiple action potentials are required to generate a calcium transient in S-neurons. How this translates to different modes of cellular depolarization and whether this is consistent across species is unknown. In this study, we used simultaneous whole-cell patch-clamp electrophysiology together with calcium imaging to investigate how enteric neurons respond to different modes of depolarization. Using both traditional (4 Hz) and also high-speed (1,000 Hz) imaging techniques, we found that single action potentials elicit calcium transients in both AH-neurons and S-neurons. Subthreshold membrane depolarizations were also able to elicit calcium transients, although calcium responses were generally amplified if an action potential was present. Furthermore, we identified that responses to nicotinic acetylcholine receptor stimulation can be used to distinguish between AH- and S-neurons in calcium imaging.NEW & NOTEWORTHY Live calcium imaging is an important tool for investigating enteric nervous system (ENS) function. Previous studies have shown that multiple action potentials are needed to generate a calcium response in S-neurons, which has important implications for the interpretation of calcium imaging data. Here, we show that in mouse myenteric neurons, calcium transients are elicited by single action potentials in both AH- and S-neurons. In addition, nicotinic acetylcholine receptor stimulation can be used to distinguish between these two classes.

A Novel Method for Identifying the Transition Zone in Long-Segment Hirschsprung Disease: Investigating the Muscle Unit to Ganglion Ratio.

Hirschsprung disease (HSCR) is characterised by the absence of enteric ganglia along variable lengths of the distal bowel. Current gold standard treatment involves the surgical resection of the defective, aganglionic bowel. Clear and reliable distinction of the normoganglionated bowel from the transition zone is key for successful resection of the entire defective bowel, and the avoidance of subsequent postoperative complications. However, the intraoperative nature of the tissue analysis and the variability of patient samples, sample preparation, and operator objectivity, make reproducible identification of the transition zone difficult. Here, we have described a novel method for using muscle units as a distinctive landmark for quantifying the density of enteric ganglia in resection specimens from HSCR patients. We show that the muscle unit to ganglion ratio is greater in the transition zone when compared with the proximal, normoganglionated region for long-segment HSCR patients. Patients with short-segment HSCR were also investigated, however, the muscle unit to ganglion ratio was not significantly different in these patients. Immunohistochemical examination of individual ganglia showed that there were no differences in the proportions of either enteric neurons or glial cells through the different regions of the resected colon. In addition, we identified that the size of enteric ganglia was smaller for patients that went on to develop HSCR associated enterocolitis; although the density of ganglia, as determined by the muscle unit to ganglia ratio, was not different when compared with patients that had no further complications. This suggests that subtle changes in the enteric nervous system, even in the "normoganglionated" colon, could be involved in changes in immune function and subsequent bacterial dysbiosis.

Surgical method to prevent early death of neonatal rat pups with Hirschsprung disease, thus permitting development of long-term therapeutic approaches.

Hirschsprung disease occurs when children are born with no intrinsic nerve cells in varying lengths of the large intestine. In the most severe cases, neurons are also missing from the distal part of the small intestine. Nerve-mediated relaxation of the aganglionic bowel fails and fecal matter accumulates in the more proximal regions of the intestine. This is life threatening. Perforation of the bowel can ensue, causing sepsis and in some cases, death of the infant. Repopulation of the colon with neural stem cells is a potential therapy, but for this to be successful the patient or experimental animal needs to survive long enough for neural precursors to differentiate and make appropriate connections. We have developed a surgical procedure that can be applied to rats with Hirschsprung disease. A stoma was created to allow the normal bowel to empty and a second stoma leading to the aganglionic bowel was also created. This allowed homozygous mutants that would usually die at less than 3 weeks of age to survive into adulthood. During this time, the rats also required post-operative care of their stomas. The interventions we describe provide an animal model of Hirschsprung disease that is suited to assess the effectiveness of cell therapies in the treatment of this condition.

Development, Diversity, and Neurogenic Capacity of Enteric Glia.

Enteric glia are a fascinating population of cells. Initially identified in the gut wall as the "support" cells of the enteric nervous system, studies over the past 20 years have unveiled a vast array of functions carried out by enteric glia. They mediate enteric nervous system signalling and play a vital role in the local regulation of gut functions. Enteric glial cells interact with other gastrointestinal cell types such as those of the epithelium and immune system to preserve homeostasis, and are perceptive to luminal content. Their functional versatility and phenotypic heterogeneity are mirrored by an extensive level of plasticity, illustrated by their reactivity in conditions associated with enteric nervous system dysfunction and disease. As one of the hallmarks of their plasticity and extending their operative relationship with enteric neurons, enteric glia also display neurogenic potential. In this review, we focus on the development of enteric glial cells, and the mechanisms behind their heterogeneity in the adult gut. In addition, we discuss what is currently known about the role of enteric glia as neural precursors in the enteric nervous system.

Development of the intrinsic innervation of the small bowel mucosa and villi.

Detection of nutritional and noxious food components in the gut is a crucial component of gastrointestinal function. Contents in the gut lumen interact with enteroendocrine cells dispersed throughout the gut epithelium. Enteroendocrine cells release many different hormones, neuropeptides, and neurotransmitters that communicate either directly or indirectly with the central nervous system and the enteric nervous system, a network of neurons and glia located within the gut wall. Several populations of enteric neurons extend processes that innervate the gastrointestinal lamina propria; however, how these processes develop and begin to transmit information from the mucosa is not fully understood. In this study, we found that Tuj1-immunoreactive neurites begin to project out of the myenteric plexus at embryonic day (E)13.5 in the mouse small intestine, even before the formation of villi. Using live calcium imaging, we discovered that neurites were capable of transmitting electrical information from stimulated villi to the plexus by E15.5. In unpeeled gut preparations where all layers were left intact, we also mimicked the basolateral release of 5-HT from enteroendocrine cells, which triggered responses in myenteric cell bodies at postnatal day (P)0. Altogether, our results show that enteric neurons extend neurites out of the myenteric plexus early during mouse enteric nervous system development, innervating the gastrointestinal mucosa, even before villus formation in mice of either sex. Neurites are already able to conduct electrical information at E15.5, and responses to 5-HT develop postnatally.NEW & NOTEWORTHY How enteric neurons project into the gut mucosa and begin to communicate with the epithelium during development is not known. Our study shows that enteric neurites project into the lamina propria as early as E13.5 in the mouse, before development of the submucous plexus and before formation of intestinal villi. These neurites are capable of transmitting electrical signals back to their cell bodies by E15.5 and respond to serotonin applied to neurite terminals by birth.

Role of JNK, MEK and adenylyl cyclase signalling in speed and directionality of enteric neural crest-derived cells.

BACKGROUND: Cells derived from the neural crest colonize the developing gut and give rise to the enteric nervous system. The rate at which the ENCC population advances along the bowel will be affected by both the speed and directionality of individual ENCCs. The aim of the study was to use time-lapse imaging and pharmacological activators and inhibitors to examine the role of several intracellular signalling pathways in both the speed and the directionality of individual enteric neural crest-derived cells in intact explants of E12.5 mouse gut. Drugs that activate or inhibit intracellular components proposed to be involved in GDNF-RET and EDN3-ETB signalling in ENCCs were used. FINDINGS: Pharmacological inhibition of JNK significantly reduced ENCC speed but did not affect ENCC directionality. MEK inhibition did not affect ENCC speed or directionality. Pharmacological activation of adenylyl cyclase or PKA (a downstream cAMP-dependent kinase) resulted in a significant decrease in ENCC speed and an increase in caudal directionality of ENCCs. In addition, adenylyl cyclase activation also resulted in reduced cell-cell contact between ENCCs, however this was not observed following PKA activation, suggesting that the effects of cAMP on adhesion are not mediated by PKA. CONCLUSIONS: JNK is required for normal ENCC migration speed, but not directionality, while cAMP signalling appears to regulate ENCC migration speed, directionality and adhesion. Collectively, our data demonstrate that intracellular signalling pathways can differentially affect the speed and directionality of migrating ENCCs.

Technologies for Live Imaging of Enteric Neural Crest-Derived Cells.

Time-lapse imaging of gut explants from embryonic mice in which neural crest-derived cells express fluorescent proteins allows the behavior of enteric neural crest cells to be observed and analyzed. Explants of embryonic gut are dissected, mounted on filter paper supports so the gut retains its tubular three-dimensional structure, and then placed in coverglass bottom culture dishes in tissue culture medium. A stainless steel ring is placed on top of the filter support to prevent movement. Imaging is performed using a confocal microscope in an environmental chamber. A z series of images through the network of fluorescent cells is collected every 3, 5, or 10 min. At the end of imaging, the z series are projected.

Regional complexity in enteric neuron wiring reflects diversity of motility patterns in the mouse large intestine.

The enteric nervous system controls a variety of gastrointestinal functions including intestinal motility. The minimal neuronal circuit necessary to direct peristalsis is well-characterized but several intestinal regions display also other motility patterns for which the underlying circuits and connectivity schemes that coordinate the transition between those patterns are poorly understood. We investigated whether in regions with a richer palette of motility patterns, the underlying nerve circuits reflect this complexity. Using Ca2+ imaging, we determined the location and response fingerprint of large populations of enteric neurons upon focal network stimulation. Complemented by neuronal tracing and volumetric reconstructions of synaptic contacts, this shows that the multifunctional proximal colon requires specific additional circuit components as compared to the distal colon, where peristalsis is the predominant motility pattern. Our study reveals that motility control is hard-wired in the enteric neural networks and that circuit complexity matches the motor pattern portfolio of specific intestinal regions.

Structurally defined signaling in neuro-glia units in the enteric nervous system.

Coordination of gastrointestinal function relies on joint efforts of enteric neurons and glia, whose crosstalk is vital for the integration of their activity. To investigate the signaling mechanisms and to delineate the spatial aspects of enteric neuron-to-glia communication within enteric ganglia we developed a method to stimulate single enteric neurons while monitoring the activity of neighboring enteric glial cells. We combined cytosolic calcium uncaging of individual enteric neurons with calcium imaging of enteric glial cells expressing a genetically encoded calcium indicator and demonstrate that enteric neurons signal to enteric glial cells through pannexins using paracrine purinergic pathways. Sparse labeling of enteric neurons and high-resolution analysis of the structural relation between neuronal cell bodies, varicose release sites and enteric glia uncovered that this form of neuron-to-glia communication is contained between the cell body of an enteric neuron and its surrounding enteric glial cells. Our results reveal the spatial and functional foundation of neuro-glia units as an operational cellular assembly in the enteric nervous system.

Publisher Correction: Kif1bp loss in mice leads to defects in the peripheral and central nervous system and perinatal death.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Optogenetic and chemogenetic techniques for neurogastroenterology.

Optogenetics and chemogenetics comprise a wide variety of applications in which genetically encoded actuators and indicators are used to modulate and monitor activity with high cellular specificity. Over the past 10 years, development of these genetically encoded tools has contributed tremendously to our understanding of integrated physiology. In concert with the continued refinement of probes, strategies to target transgene expression to specific cell types have also made much progress in the past 20 years. In addition, the successful implementation of optogenetic and chemogenetic techniques thrives thanks to ongoing advances in live imaging microscopy and optical technology. Although innovation of optogenetic and chemogenetic methods has been primarily driven by researchers studying the central nervous system, these techniques also hold great promise to boost research in neurogastroenterology. In this Review, we describe the different classes of tools that are currently available and give an overview of the strategies to target them to specific cell types in the gut wall. We discuss the possibilities and limitations of optogenetic and chemogenetic technology in the gut and provide an overview of their current use, with a focus on the enteric nervous system. Furthermore, we suggest some experiments that can advance our understanding of how the intrinsic and extrinsic neural networks of the gut control gastrointestinal function.

Kif1bp loss in mice leads to defects in the peripheral and central nervous system and perinatal death.

Goldberg-Shprintzen syndrome is a poorly understood condition characterized by learning difficulties, facial dysmorphism, microcephaly, and Hirschsprung disease. GOSHS is due to recessive mutations in KIAA1279, which encodes kinesin family member 1 binding protein (KIF1BP, also known as KBP). We examined the effects of inactivation of Kif1bp in mice. Mice lacking Kif1bp died shortly after birth, and exhibited smaller brains, olfactory bulbs and anterior commissures, and defects in the vagal and sympathetic innervation of the gut. Kif1bp was found to interact with Ret to regulate the development of the vagal innervation of the stomach. Although newborn Kif1bp -/- mice had neurons along the entire bowel, the colonization of the gut by neural crest-derived cells was delayed. The data show an essential in vivo role for KIF1BP in axon extension from some neurons, and the reduced size of the olfactory bulb also suggests additional roles for KIF1BP. Our mouse model provides a valuable resource to understand GOSHS.