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Scientific Reports Feb 2024The enteric nervous system (ENS) is a large and complex part of the peripheral nervous system, and it is vital for gut homeostasis. To study the ENS, different hyper-...
The enteric nervous system (ENS) is a large and complex part of the peripheral nervous system, and it is vital for gut homeostasis. To study the ENS, different hyper- and hypo-innervated model systems have been developed. The NSE-Noggin mouse model was described as one of the few models with a higher enteric neuronal density in the colon. However, in our hands NSE-Noggin mice did not present with a hyperganglionic phenotype. NSE-Noggin mice were phenotyped based on fur appearance, genotyped and DNA sequenced to demonstrate transgene and intact NSE-Noggin-IRES-EGFP construct presence, and RNA expression of Noggin was shown to be upregulated. Positive EGFP staining in the plexus of NSE-Noggin mice also confirmed Noggin protein expression. Myenteric plexus preparations of the colon were examined to quantify both the overall density of enteric neurons and the proportions of enteric neurons expressing specific subtype markers. The total number of enteric neurons in the colonic myenteric plexus of transgenic mice did not differ significantly from wild types, nor did the proportion of calbindin, calretinin, or serotonin immunoreactive myenteric neurons. Possible reasons as to why the hyperinnervated phenotype could not be observed in contrast with original studies using this mouse model are discussed, including study design, influence of microbiota, and other environmental variables.
Topics: Mice; Animals; Neurons; Enteric Nervous System; Carrier Proteins; Myenteric Plexus; Mice, Transgenic; Colon
PubMed: 38355947
DOI: 10.1038/s41598-024-54337-w -
Frontiers in Immunology 2024Wnt-signaling is a key regulator of stem cell homeostasis, extensively studied in the intestinal crypt and other metazoan tissues. Yet, there is hardly any data...
INTRODUCTION
Wnt-signaling is a key regulator of stem cell homeostasis, extensively studied in the intestinal crypt and other metazoan tissues. Yet, there is hardly any data available on the presence of Wnt-signaling components in the adult enteric nervous system (ENS) .
METHODS
Therefore, we employed RNAscope HiPlex-assay, a novel and more sensitive hybridization technology. By amplifying target specific signals, this technique enables the detection of low abundance, tightly regulated RNA content as is the case for Wnt-signaling components. Additionally, we compared our data to previously published physiological single cell RNA and RiboTag-based RNA sequencing analyses of enteric gliosis using data-mining approaches.
RESULTS
Our descriptive analysis shows that several components of the multidi-mensional regulatory network of the Wnt-signaling pathway are present in the murine ENS. The transport and secretion protein for Wnt-ligands Wntless as well as canonical (Wnt3a and Wnt2b) and non-canonical Wnt-ligands (Wnt5a, Wnt7a, Wnt8b and Wnt11) are detectable within submucosal and myenteric plexus. Further, corresponding Frizzled receptors (Fzd1, Fzd3, Fzd6, and Fzd7) and regulatory signaling mediators like R-Spondin/DKK ligands are present in the ENS of the small and large intestine. Further, data mining approaches revealed, that several Wnt-related molecules are expressed by enteric glial cell clusters and are dynamically regulated during the inflammatory manifestation of enteric gliosis.
DISCUSSION
Our results suggest, that canonical and non-canonical Wnt-signaling has a much broader impact on the mature ENS and its cellular homeostasis in health and inflammation, than previously anticipated.
Topics: Animals; Mice; Transcriptome; Gliosis; Wnt Signaling Pathway; Enteric Nervous System; Ligands; RNA
PubMed: 38322254
DOI: 10.3389/fimmu.2024.1302488 -
Frontiers in Neuroscience 2023Viscerofugal neurons (VFNs) have cell bodies in the myenteric plexus and axons that project to sympathetic prevertebral ganglia. In animals they activate sympathetic...
BACKGROUND AND AIMS
Viscerofugal neurons (VFNs) have cell bodies in the myenteric plexus and axons that project to sympathetic prevertebral ganglia. In animals they activate sympathetic motility reflexes and may modulate glucose metabolism and feeding. We used rapid retrograde tracing from colonic nerves to identify VFNs in human colon for the first time, using preparations with multi-layer immunohistochemistry.
METHODS
Colonic nerves were identified in isolated preparations of human colon and set up for axonal tracing with biotinamide. After fixation, labeled VFN cell bodies were subjected to multiplexed immunohistochemistry for 12 established nerve cell body markers.
RESULTS
Biotinamide tracing filled 903 viscerofugal nerve cell bodies ( = 23), most of which (85%) had axons projecting orally before entering colonic nerves. Morphologically, 97% of VFNs were uni-axonal. Of 215 VFNs studied in detail, 89% expressed ChAT, 13% NOS, 13% calbindin, 9% enkephalin, 7% substance P and 0 of 123 VFNs expressed CART. Few VFNs contained calretinin, VIP, 5HT, CGRP, or NPY. VFNs were often surrounded by dense baskets of axonal varicosities, probably reflecting patterns of connectivity; VAChT+ (cholinergic), SP+ and ENK+ varicosities were most abundant around them. Human VFNs were diverse; showing 27 combinations of immunohistochemical markers, 4 morphological types and a wide range of cell body sizes. However, 69% showed chemical coding, axonal projections, soma-dendritic morphology and connectivity similar to enteric excitatory motor neurons.
CONCLUSION
Viscerofugal neurons are present in human colon and show very diverse combinations of features. High proportions express ChAT, consistent with cholinergic synaptic outputs onto postganglionic sympathetic neurons in prevertebral ganglia.
PubMed: 38292899
DOI: 10.3389/fnins.2023.1313057 -
International Journal of Molecular... Jan 2024The brain-gut axis has been identified as an important contributor to the physiopathology of Parkinson's disease. In this pathology, inflammation is thought to be driven... (Review)
Review
The brain-gut axis has been identified as an important contributor to the physiopathology of Parkinson's disease. In this pathology, inflammation is thought to be driven by the damage caused by aggregation of α-synuclein in the brain. Interestingly, the Braak's theory proposes that α-synuclein misfolding may originate in the gut and spread in a "prion-like" manner through the vagus nerve into the central nervous system. In the enteric nervous system, enteric glial cells are the most abundant cellular component. Several studies have evaluated their role in Parkinson's disease. Using samples obtained from patients, cell cultures, or animal models, the studies with specific antibodies to label enteric glial cells (GFAP, Sox-10, and S100β) seem to indicate that activation and reactive gliosis are associated to the neurodegeneration produced by Parkinson's disease in the enteric nervous system. Of interest, Toll-like receptors, which are expressed on enteric glial cells, participate in the triggering of immune/inflammatory responses, in the maintenance of intestinal barrier integrity and in the configuration of gut microbiota; thus, these receptors might contribute to Parkinson's disease. External factors like stress also seem to be relevant in its pathogenesis. Some authors have studied ways to reverse changes in EGCs with interventions such as administration of Tryptophan-2,3-dioxygenase inhibitors, nutraceuticals, or physical exercise. Some researchers point out that beyond being activated during the disease, enteric glial cells may contribute to the development of synucleinopathies. Thus, it is still necessary to further study these cells and their role in Parkinson's disease.
Topics: Animals; Humans; Parkinson Disease; alpha-Synuclein; Brain; Inflammation; Neuroglia; Enteric Nervous System
PubMed: 38279293
DOI: 10.3390/ijms25021294 -
Scientific Reports Jan 2024Achalasia is a rare esophageal motility disorder for which the etiology is not fully understood. Evidence suggests that autoimmune inflammatory infiltrates, possibly...
Achalasia is a rare esophageal motility disorder for which the etiology is not fully understood. Evidence suggests that autoimmune inflammatory infiltrates, possibly triggered by a viral infection, may lead to a degeneration of neurons within the myenteric plexus. While the infection is eventually resolved, genetically susceptible individuals may still be at risk of developing achalasia. This study aimed to determine whether immunological and physiological networks differ between male and female patients with achalasia. This cross-sectional study included 189 preoperative achalasia patients and 500 healthy blood donor volunteers. Demographic, clinical, laboratory, immunological, and tissue biomarkers were collected. Male and female participants were evaluated separately to determine the role of sex. Correlation matrices were constructed using bivariate relationships to generate complex inferential networks. These matrices were filtered based on their statistical significance to identify the most relevant relationships between variables. Network topology and node centrality were calculated using tools available in the R programming language. Previous occurrences of chickenpox, measles, and mumps infections have been proposed as potential risk factors for achalasia, with a stronger association observed in females. Principal component analysis (PCA) identified IL-22, Th2, and regulatory B lymphocytes as key variables contributing to the disease. The physiological network topology has the potential to inform whether a localized injury or illness is likely to produce systemic consequences and the resulting clinical presentation. Here we show that immunological involvement in achalasia appears localized in men because of their highly modular physiological network. In contrast, in women the disease becomes systemic because of their robust network with a larger number of inter-cluster linkages.
Topics: Humans; Female; Male; Esophageal Achalasia; Cross-Sectional Studies; Esophageal Motility Disorders; B-Lymphocytes, Regulatory; Blood Donors
PubMed: 38267468
DOI: 10.1038/s41598-024-52273-3 -
International Journal of Molecular... Jan 2024Recent discoveries have shown that enteric glial cells play an important role in different neurodegenerative disorders, such as Parkinson's disease (PD), which is...
Recent discoveries have shown that enteric glial cells play an important role in different neurodegenerative disorders, such as Parkinson's disease (PD), which is characterized by motor dysfunctions caused by the progressive loss of dopaminergic neurons in the substance nigra pars compacta and non-motor symptoms including gastrointestinal dysfunction. In this study, we investigated the modulatory effects of the flavonoid rutin on the behavior and myenteric plexuses in a PD animal model and the response of enteric glia. Adult male Wistar rats were submitted to stereotaxic injection with 6-hydroxydopamine or saline, and they were untreated or treated with rutin (10 mg/kg) for 14 days. The ileum was collected to analyze tissue reactivity and immunohistochemistry for neurons (HuC/HuD) and enteric glial cells (S100β) in the myenteric plexuses. Behavioral tests demonstrated that treatment with rutin improved the motor capacity of parkinsonian animals and improved intestinal transit without interfering with the cell population; rutin treatment modulated the reactivity of the ileal musculature through muscarinic activation, reducing relaxation through the signaling pathway of nitric oxide donors, and increased the longitudinal contractility of the colon musculature in parkinsonian animals. Rutin revealed modulatory activities on the myenteric plexus, bringing relevant answers regarding the effect of the flavonoid in this system and the potential application of PD adjuvant treatment.
Topics: Male; Rats; Animals; Rats, Wistar; Myenteric Plexus; Flavonoids; Rutin; Parkinson Disease; Disease Models, Animal; Dopaminergic Neurons
PubMed: 38256111
DOI: 10.3390/ijms25021037 -
International Journal of Molecular... Dec 2023Bacteria in the gut microbiome play an intrinsic part in immune activation, intestinal permeability, enteric reflex, and entero-endocrine signaling. The gut microbiota... (Review)
Review
Bacteria in the gut microbiome play an intrinsic part in immune activation, intestinal permeability, enteric reflex, and entero-endocrine signaling. The gut microbiota communicates with the central nervous system (CNS) through the production of bile acids, short-chain fatty acids (SCFAs), glutamate (Glu), γ-aminobutyric acid (GABA), dopamine (DA), norepinephrine (NE), serotonin (5-HT), and histamine. A vast number of signals generated in the gastrointestinal tract (GIT) reach the brain via afferent fibers of the vagus nerve (VN). Signals from the CNS are returned to entero-epithelial cells (EES) via efferent VN fibers and communicate with 100 to 500 million neurons in the submucosa and myenteric plexus of the gut wall, which is referred to as the enteric nervous system (ENS). Intercommunications between the gut and CNS regulate mood, cognitive behavior, and neuropsychiatric disorders such as autism, depression, and schizophrenia. The modulation, development, and renewal of nerves in the ENS and changes in the gut microbiome alter the synthesis and degradation of neurotransmitters, ultimately influencing our mental health. The more we decipher the gut microbiome and understand its effect on neurotransmission, the closer we may get to developing novel therapeutic and psychobiotic compounds to improve cognitive functions and prevent mental disorders. In this review, the intricate control of entero-endocrine signaling and immune responses that keep the gut microbiome in a balanced state, and the influence that changing gut bacteria have on neuropsychiatric disorders, are discussed.
Topics: Humans; Mental Health; Gastrointestinal Microbiome; Central Nervous System; Enteric Nervous System; Glutamic Acid
PubMed: 38203207
DOI: 10.3390/ijms25010038 -
American Journal of Physiology.... May 2024The 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 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. 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.
Topics: Neurons; Enteric Nervous System; Myenteric Plexus; Submucous Plexus
PubMed: 38193168
DOI: 10.1152/ajpgi.00170.2023 -
BMC Gastroenterology Jan 2024This study was designed to explore the expression changes of P2Y receptors in the distal colonic myenteric layer of rats. An opioid induced constipation(OIC) rat model...
This study was designed to explore the expression changes of P2Y receptors in the distal colonic myenteric layer of rats. An opioid induced constipation(OIC) rat model was generated by intraperitoneal (i.p) injection of loperamide. At 7 days post-treatment, the model rats were assessed by calculating the fecal water content and the gastrointestinal transit ratio. The immunofluorescence (IF)-based histochemical study was used to observe the distribution of P2Y receptors in the distal colonic myenteric plexus. Western blotting (WB) was performed to evaluate the expression changes of P2Y proteins in the myenteric layer, and the electrophysiological approaches were carried out to determine the regulatory roles of P2Y receptors on distal colonic motor function. IF showed that P2Y receptors are co-expressed MOR in the enteric nerve cells of the distal colonic myenteric plexus. Moreover, the WB revealed that the protein levels of P2Y were significantly decreased in the distal colonic myenteric layer of OIC rats. In vitro tension experiments exhibited that the P2Y receptor antagonist MRS2500 enhanced the spontaneous contraction amplitude, adding EM2 and β-FNA did not have any effect on MRS2500. Therefore, P2Y receptor expression could be associated with the occurrence of OIC in this rat model and the regulation of colonic motility by MOR may be related to the release of purine neurotransmitters such as ATP in the colonic nervous system.
Topics: Animals; Rats; Myenteric Plexus; Analgesics, Opioid; Opioid-Induced Constipation; Constipation; Blotting, Western
PubMed: 38191294
DOI: 10.1186/s12876-024-03119-9 -
Animals : An Open Access Journal From... Dec 2023In the expansive domain of neuropeptide investigation, spexin (SPX) has emerged as a captivating subject, exerting a significant impact on diverse physiological...
In the expansive domain of neuropeptide investigation, spexin (SPX) has emerged as a captivating subject, exerting a significant impact on diverse physiological processes. Initially identified in mice, SPX's distribution transcends various organs, suggesting its potential regulatory roles. Despite extensive research in smaller species, a notable gap exists in our comprehension of SPX in larger mammals, particularly ruminants. Our study meticulously explores the immunolocalization of SPX within the gastrointestinal organs of bovines, with a specific focus on the abomasum, jejunum, and colon. Tissue samples from Holstein-Friesian cattle underwent careful processing, and gene mRNA expression levels, particularly and , were assessed. Intriguingly, our findings revealed that expression was highest in the jejunum, signifying a potentially critical role in this digestive segment. Immunohistochemistry further unveiled distinct patterns of SPX immunoreactivity in each examined region-abomasum, jejunum, and colon-highlighting nuanced, region-specific responses. Notably, the abomasum and jejunum predominantly exhibited positive immunoreactivity in the submucosal plexus, while the colon, in contrast, demonstrated a higher degree of immunoreactivity in myenteric plexus neurons. Our investigation, grounded in the hypothesis of ubiquitous SPX distribution in ruminants, delves deeper into the intricate role of SPX within the enteric nervous system. This study meticulously explores the spatial distribution of SPX within the myenteric and submucosal plexuses, integral components of the enteric nervous system. These findings significantly enhance our understanding of SPX's potential roles in gastrointestinal regulation in bovines, providing a unique perspective on larger mammals and enriching our comprehension of this intriguing neuropeptide's significance in various physiological processes.
PubMed: 38136826
DOI: 10.3390/ani13243789