Myenteric Neurons Research Articles

Atropine-functionalized gold nanoparticles binding to muscarinic receptors after passage across the intestinal epithelium.

Gold nanoparticles have a high potential to be a treatment of diseases by their specific drug delivery properties and multivalent receptor stimulation. For the present project, spherical gold nanoparticles were synthesized and functionalized with the muscarinic receptor antagonist atropine (Au-MUDA-AT NPs). The diameter of the gold core could precisely be controlled by using different synthetic methods and reducing agents resulting in functionalized gold nanoparticles with diameters ranging from 8 to 16 nm. The ability to interact with intestinal muscarinic receptors is size-dependent. When using intestinal chloride secretion induced by the stable acetylcholine derivative, carbachol, as read-out, the strongest inhibition, i.e. the most efficient blockade of muscarinic receptors, was observed with 13 nm sized Au-MUDA-AT NPs. Functional experiments indicate that Au-MUDA-AT NPs with a diameter of 14 nm are able to pass the intestinal mucosa in a time-dependent manner after administration to the intestinal lumen. For example, luminally administered Au-MUDA-AT NPs inhibited contractions of the small intestinal longitudinal muscle layer induced by electrical stimulation of myenteric neurons. A similar inhibition of basolateral epithelial receptors was observed after luminal administration of Au-MUDA-AT NPs when using carbachol-induced chloride secretion across the intestinal epithelium as a test system. Thus, Au-MUDA-AT NPs might be a therapeutic tool for the modulation of intestinal secretion and motility after oral application in the future.

S2266 Achalasia as a Sequelae of COVID-19

Introduction: Achalasia is a neurodegenerative motility disorder of distal esophagus and lower esophageal sphincter characterized by loss of myenteric neurons in esophagus. It therefore results in deranged peristalsis and characteristic manifestations of regurgitation, dysphagia and retrosternal chest pressure sensation. Recent studies have suggested inflammatory neurodegenerative insult from viral infection as a potential etiology. Sars-Cov-2 causes multiple gastrointestinal (GI) manifestations like ageusia, nausea, vomiting, pain and diarrhea. We present a rare case of achalasia that presented soon after COVID infection in an otherwise healthy male. Case Description/Methods: A 63-year-old man was referred for further evaluation of dysphagia that started a few months after a non-severe COVID infection with associated ageusia. He reported progressive dysphagia to both solids and liquids with concomitant regurgitation and chest pain. By the time he presented, he had lost 45 lbs over the course of 6 months and had an Eckardt score of 8. High resolution manometry was suggestive of evolving type II achalasia. A follow up Endoflip exam with a 16 cm catheter inflated to 60 cc revealed a esophagogastric diameter of 7.3 mm and a distensibility index (DI) of 1.9. Timed barium esophagogram showed a 5 cm contrast column in distal esophagus at 5 minutes. The patient has since been referred for per oral endoscopic myotomy (Figure). Discussion: Sars-Cov-2 enters the GI epithelial cells through ACE2 receptors and exerts its effects through an aberrant immune response involving T- lymphocytes rather than direct epithelial damage. An anomalous immune reaction resulting in degeneration of inhibitory neurons in the myenteric plexus may be the cause of achalasia in COVID-19. On literature review, we came across at least 1 case report of a patient developing achalasia after COVID-19. A Venezuelan study noted that the frequency of patients with achalasia during the year 2020 and 2021 was far greater than those of previous years and at least 2/3rd of these patients had presented with COVID-19 infection. It will be interesting to note if coronavirus can be isolated from muscle biopsies of these subjects as has been the case with VZV previously. Further investigation especially into patients presenting with GI symptoms with COVID infection is warranted.Figure 1.: Endoflip ; Barium Esophageogram.

Neonatal antibiotics have long term sex-dependent effects on the enteric nervous system.

Infants and young children receive the highest exposures to antibiotics globally. Although there is building evidence that early life exposure to antibiotics increases susceptibility to various diseases including gut disorders later in life, the lasting impact of early life antibiotics on the physiology of the gut and its enteric nervous system (ENS) remains unclear. We treated neonatal mice with the antibiotic vancomycin during their first 10 postnatal days, then examined potential lasting effects of the antibiotic treatment on their colons during young adulthood (6weeks old). We found that neonatal vancomycin treatment disrupted the gut functions of young adult female and male mice differently. Antibiotic-exposed females had significantly longer whole gut transit while antibiotic-treated males had significantly lower faecal weights compared to controls. Both male and female antibiotic-treated mice had greater percentages of faecal water content. Neonatal vancomycin treatment also had sexually dimorphic impacts on the neurochemistry and Ca2+ activity of young adult myenteric and submucosal neurons. Myenteric neurons of male mice were more disrupted than those of females, while opposing changes in submucosal neurons were seen in each sex. Neonatal vancomycin also induced sustained changes in colonic microbiota and lasting depletion of mucosal serotonin (5-HT) levels. Antibiotic impacts on microbiota and mucosal 5-HT were not sex-dependent, but we propose that the responses of the host to these changes are sex-specific. This first demonstration of long-term impacts of neonatal antibiotics on the ENS, gut microbiota and mucosal 5-HT has important implications for gut function and other physiological systems of the host. KEY POINTS: Early life exposure to antibiotics can increase susceptibility to diseases including functional gastrointestinal (GI) disorders later in life. Yet, the lasting impact of this common therapy on the gut and its enteric nervous system (ENS) remains unclear. We investigated the long-term impact of neonatal antibiotic treatment by treating mice with the antibiotic vancomycin during their neonatal period, then examining their colons during young adulthood. Adolescent female mice given neonatal vancomycin treatment had significantly longer whole gut transit times, while adolescent male and female mice treated with neonatal antibiotics had significantly wetter stools. Effects of neonatal vancomycin treatment on the neurochemistry and Ca2+ activity of myenteric and submucosal neurons were sexually dimorphic. Neonatal vancomycin also had lasting effects on the colonic microbiome and mucosal serotonin biosynthesis that were not sex-dependent. Different male and female responses to antibiotic-induced disruptions of the ENS, microbiota and mucosal serotonin biosynthesis can lead to sex-specific impacts on gut function.

Quantification of CGRP-immunoreactive myenteric neurons in mouse colon.

Quantitative data of biological systems provide valuable baseline information for understanding pathology, experimental perturbations, and computational modeling. In mouse colon, calcitonin gene-related peptide (CGRP) is expressed by myenteric neurons with multiaxonal (Dogiel type II) morphology, characteristic of intrinsic primary afferent neurons (IPANs). Analogous neurons in other species and gut regions represent 5-35% of myenteric neurons. We aimed to quantify proportions of CGRP-immunopositive (CGRP+) myenteric neurons. Colchicine-treated wholemount preparations of proximal, mid, and distal colon were labeled for HuC/D, CGRP, nitric oxide synthase (NOS), and peripherin (Per). The pan-neuronal markers (Hu+/Per+) co-labeled 94% of neurons. Hu+/Per- neurons comprised ∼6%, but Hu-/Per+ cells were rare. Thus, quantification was based on Hu+ myenteric neurons (8576 total; 1225±239 per animal, n=7). CGRP+ cell bodies were significantly larger than the average of all Hu+ neurons (329±13 vs. 261±12μm2 , p<.0001). CGRP+ neurons comprised 19%±3% of myenteric neurons without significant regional variation. NOS+ neurons comprised 42%±2% of myenteric neurons overall, representing a lower proportion in proximal colon, compared to mid and distal colon (38%±2%, 44%±2%, and 44%±3%, respectively). Peripherin immunolabeling revealed cell body and axonal morphology in some myenteric neurons. Whether all CGRP+ neurons were multiaxonal could not be addressed using peripherin immunolabeling. However, of 118 putatively multiaxonal neurons first identified based on peripherin immunoreactivity, all were CGRP+ (n=4). In conclusion, CGRP+ myenteric neurons in mouse colon were comprehensively quantified, occurring within a range expected of a putative IPAN marker. All Per+ multiaxonal neurons, characteristic of Dogiel type II/IPAN morphology, were CGRP+.

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.

P2X7 receptor blockade decreases inflammation, apoptosis, and enteric neuron loss during Clostridioides difficile toxin A-induced ileitis in mice.

BACKGROUND Clostridioides difficile (C. difficile) is the most common pathogen causing health care-associated infections. C. difficile TcdA and TcdB have been shown to activate enteric neurons; however, what population of these cells is more profoundly influenced and the mechanism underlying these effects remain unknown.AIMTo characterize a specific population of TcdA-affected myenteric neurons and investigate the role of the P2X7 receptor in TcdA-induced ileal inflammation, cell death, and the changes in the enteric nervous system in mice.METHODSSwiss mice were used to model TcdA-induced ileitis in ileal loops exposed to TcdA (50 μg/Loop) for 4 h. To investigate the role of the P2X7 receptor, Brilliant Blue G (50 mg/kg, i.p.), which is a nonspecific P2X7 receptor antagonist, or A438079 (0.7 μg/mouse, i.p.), which is a competitive P2X7 receptor antagonist, were injected one hour prior to TcdA challenge. Ileal samples were collected to analyze the expression of the P2X7 receptor (by quantitative real-time polymerase chain reaction and immunohistochemistry), the population of myenteric enteric neurons (immunofluorescence), histological damage, intestinal inflammation, cell death (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling), neuronal loss, and S100B synthesis (immunohistochemistry).RESULTSTcdA upregulated (P < 0.05) the expression of the P2X7 receptor gene in the ileal tissues, increasing the level of this receptor in myenteric neurons compared to that in control mice. Comparison with the control mice indicated that TcdA promoted (P < 0.05) the loss of myenteric calretinin+ (Calr) and choline acetyltransferase+ neurons and increased the number of nitrergic+ and Calr+ neurons expressing the P2X7 receptor. Blockade of the P2X7 receptor decreased TcdA-induced intestinal damage, cytokine release [interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α], cell death, enteric neuron loss, and S100B synthesis in the mouse ileum.CONCLUSIONOur findings demonstrated that TcdA induced the upregulation of the P2X7 receptor, which promoted enteric neuron loss, S100B synthesis, tissue damage, inflammation, and cell death in the mouse ileum. These findings contribute to the future directions in understanding the mechanism involved in intestinal dysfunction reported in patients after C. difficile infection.

Neurochemical classification of serotonin-immunoreactive neurons co-innervating motor endplates in the mouse esophagus.

Serotonin immunoreactivity was previously found in myenteric neurons co-innervating motor endplates in the mouse esophagus striated muscle and an involvement in motility control was suggested. However, it is not known if other neuroactive substances are present in these neurons and to what extent they co-localize. First, vasoactive intestinal peptide (VIP) was established as a bona fide marker for putative inhibitory myenteric neurons by evaluating co-localization with neuronal nitric oxide synthase (nNOS) and neuropeptide Y (NPY). Then, co-localization of serotonin and VIP was tested in co-innervating axons on motor endplates, which were visualized with α-bungarotoxin (α-BT) by multilabel immunofluorescence. Myenteric ganglia were also surveyed for co-localization in neuronal perikarya and varicosities. nNOS, NPY, and VIP were completely co-localized in enteric co-innervating nerve terminals on motor endplates. After co-staining with VIP, we found (a) serotonin (5-HT)-positive nerve endings without VIP (44% of 5-HT-positively innervated endplates), (b) 5-HT- and VIP-positive endings without co-localization (35%), and (c) 5-HT- and VIP-positive endings with co-localization (21%). About one-fifth of nerve terminals on motor endplates containing 5-HT originate from putative inhibitory peptidegic nitrergic neurons. However, the majority represents a different population presumably subserving different functions.

Histological and neuronal changes in the duodenum of hamsters infected with Leishmania (Leishmania) infantum

Visceral leishmaniasis is a neglected tropical disease caused by parasites belonging to the Leishmania genus that infect macrophages in different tissues such as the spleen, liver, lymph nodes, bone marrow, and intestine. Therefore, this study aimed to investigate the integrity of the intestinal tract and the nitrergic (NADPH-dp) and metabolically active (NADH-dp) myenteric neurons of the duodenum of golden hamsters infected with L. (L.) infantum. Therefore, thirty golden hamsters were divided into six groups (n = 5); three of them were infected with 2 × 107 promastigote forms of L. (L.) infantum by intraperitoneal route (Infected Group - IG) and three were inoculated with saline solution (control group - CG). After 30, 60 and 90 days post-infection (DPI) infected animals were euthanized and the liver, spleen and duodenum were collected to analyze tissue parasitism. The duodenum was processed using usual histological techniques to analyze the main changes that occurred during infection and histochemical techniques to phenotype myenteric neurons. Amastigote forms were observed in the spleen, liver, and duodenum during all experimental periods, and tissue parasitism in these organs increased significantly over time. At 30 DPI, reduction in muscle tunic, increase in the total intestinal wall and the number of goblet cells PAS+ was observed. At 60 DPI, an increase in intestinal crypts and intraepithelial lymphocytes was observed, and a reduction in intestinal villi was observed at 90 DPI, along with an increase in crypt size. Regarding neurons, an increase in the density of the NADPH-dp population was observed at 30 DPI, but at 60 and 90 DPI a significant reduction of this population was observed. In general, infection progression was observed to cause significant morphofunctional changes in the duodenum of infected hamsters.

Morphology of human fetal enteric neurons: A comparative study of different segments of the colon

ObjectivesThe Enteric Nervous System (ENS) present in the wall of the gut is currently being explored because of its influence on the gut and beyond. In this context, the morphology of developing ENS has not been completely understood in humans due to lack of adequate literature. The aim of the present study was to observe the morphology of the enteric neurons in the human fetal colon and compare the findings in ascending colon a midgut derivative and descending colon a hindgut derivative at various weeks of gestation (WG). Material and methodsTissue samples from 15 aborted fetuses (11 WG to 2 months postnatal) were processed for Cresyl violet, H & E staining, and NADPH Diaphorase histochemistry. The morphometric analysis was done by calculating the neuronal number density and neuronal fractional area. The Student t-test; Mann–Whitney test and Wilcoxon signed-rank test were used to analyze the data. ResultsThe muscularis externa with two distinct layers was visible as early as 13 WG and the muscularis mucosae was first observed at 18 WG. The size of the myenteric neurons appeared to be larger with increasing weeks of gestation suggesting a process of neuronal maturation. The neuronal number density and neuronal fractional area seemed to be reduced with advancing fetal age. There was no marked difference between the ascending and sigmoid colon. At 23 and 26 WG, a mature pattern of nitrergic innervation was observed. ConclusionThis study is done on human fetal tissue samples unlike previous studies on animal samples to comprehend the morphology of developing ENS. It will aid in understanding the effect of ENS on various neurological disorders.

Dysregulation of the Enteric Nervous System in the Mid Colon of Complement Component 3 Knockout Mice with Constipation Phenotypes.

Complement component 3 (C3) contributes to neurogenesis, neural migration, and synaptic elimination under normal and disease conditions of the brain, even though it has not been studied in the enteric nervous system (ENS). To determine the role of C3 in the regulatory mechanism of ENS during C3 deficiency-induced constipation, the changes in the markers of neuronal and interstitial cells of Cajal (ICCs), the markers for excitatory and inhibitory transmission of ENS, and expression of C3 receptors were analyzed in the mid colon of C3 knockout (KO) mice at 16 weeks of age. Prominent constipation phenotypes, including the decrease in stool parameters, changes in the histological structure, and suppression of mucin secretion, were detected in C3 KO mice compared to wildtype (WT) mice. The expression levels of the neuron specific enolase (NSE), protein gene product 9.5 (PGP9.5), and C-kit markers for myenteric neurons and ICCs were lower in the mid colon of C3 KO mice than WT mice. Excitatory transmission analysis revealed similar suppression of the 5-hydroxytryptamine (5-HT) concentration, expression of 5-HT receptors, acetylcholine (ACh) concentration, ACh esterase (AChE) activity, and expression of muscarinic ACh receptors (mAChRs), despite the mAChRs downstream signaling pathway being activated in the mid colon of C3 KO mice. In inhibitory transmission analysis, C3 KO mice showed an increase in the nitric oxide (NO) concentration and inducible nitric oxide synthase (iNOS) expression, while neuronal NOS (nNOS) expression, cholecystokinin (CCK), and gastrin concentration were decreased in the same mice. Furthermore, the levels of C3a receptor (C3aR) and C3bR expression were enhanced in the mid colon of C3 KO mice compared to the WT mice during C3 deficiency-induced constipation. Overall, these results indicate that a dysregulation of the ENS may play an important role in C3 deficiency-induced constipation in the mid colon of C3 KO mice.

Neurochemical profile of the myenteric plexus in the rat colorectal region.

The enteric nervous system, a major subdivision of the autonomic nervous system, is known for its neurochemical heterogeneity and complexity. The myenteric plexus, one of its two principal components, primarily controls peristalsis and its dysfunction may lead to a number of gastrointestinal motility disorders. The myenteric neurons have been described to use a wide variety of neurotransmitters although no evidence has been reported for the existence of adrenergic neurons in the hindgut. This study aims at elucidating the chemical coding of neurons in the myenteric plexus of the rat colon and anorectal region with particular emphasis on cholinergic and the so-called nonadrenergic, noncholinergic (NANC) transmitter systems. The immunostaining for choline acetyltransferase revealed an intense staining of the myenteric ganglia with clear delineation of their neuronal cell bodies and without local distributional differences in the colonic region. The myenteric ATPergic structures were mostly limited to fiber bundles surrounding unstained myenteric neurons and penetrating the two muscle layers. We also observed an abundance of intensely stained varicose substance P-immunopositive fibers, ensheathing the immunonegative myenteric neuronal cell bodies in a basket-like manner. Applying NADPH-diaphorase histochemistry and nitric oxide synthase immunohistochemistry, we were able to demonstrate numerous nitrergic somata of myenteric neurons with Dogiel Type I morphology. Apart from the observed nitrergic distributional patterns, no distinct variations were found in the staining intensity or distribution of myenteric structures in the colon and anorectal area. Our results suggest that myenteric neurons in the distal intestinal portion utilize a broad spectrum of enteric transmitters, including classical and NANC transmitters.

Neuroprotective Effects of VEGF in the Enteric Nervous System.

Although the enteric nervous system (ENS) functions largely autonomously as part of the peripheral nervous system (PNS), it is connected to the central nervous system (CNS) via the gut–brain axis. In many neurodegenerative diseases, pathological changes occur in addition to gastrointestinal symptoms, such as alpha-synuclein aggregates in Parkinson’s disease, which are found early in the ENS. In both the CNS and PNS, vascular endothelial growth factor (VEGF) mediates neuroprotective and neuroregenerative effects. Since the ENS with its close connection to the microbiome and the immune system is discussed as the origin of neurodegenerative diseases, it is necessary to investigate the possibly positive effects of VEGF on enteric neurons. Using laser microdissection and subsequent quantitative RT-PCR as well as immunohistochemistry, for the first time we were able to detect and localize VEGF receptor expression in rat myenteric neurons of different ages. Furthermore, we demonstrate direct neuroprotective effects of VEGF in the ENS in cell cultures. Thus, our results suggest a promising approach regarding neuroprotection, as the use of VEGF (may) prevent neuronal damage in the ENS.

Computational simulations and Ca2+ imaging reveal that slow synaptic depolarizations (slow EPSPs) inhibit fast EPSP evoked action potentials for most of their time course in enteric neurons.

Transmission between neurons in the extensive enteric neural networks of the gut involves synaptic potentials with vastly different time courses and underlying conductances. Most enteric neurons exhibit fast excitatory post-synaptic potentials (EPSPs) lasting 20-50 ms, but many also exhibit slow EPSPs that last up to 100 s. When large enough, slow EPSPs excite action potentials at the start of the slow depolarization, but how they affect action potentials evoked by fast EPSPs is unknown. Furthermore, two other sources of synaptic depolarization probably occur in enteric circuits, activated via GABAA or GABAC receptors; how these interact with other synaptic depolarizations is also unclear. We built a compartmental model of enteric neurons incorporating realistic voltage-dependent ion channels, then simulated fast EPSPs, slow EPSPs and GABAA or GABAC ligand-gated Cl- channels to explore these interactions. Model predictions were tested by imaging Ca2+ transients in myenteric neurons ex vivo as an indicator of their activity during synaptic interactions. The model could mimic firing of myenteric neurons in mouse colon evoked by depolarizing current during intracellular recording and the fast and slow EPSPs in these neurons. Subthreshold fast EPSPs evoked spikes during the rising phase of a slow EPSP, but suprathreshold fast EPSPs could not evoke spikes later in a slow EPSP. This predicted inhibition was confirmed by Ca2+ imaging in which stimuli that evoke slow EPSPs suppressed activity evoked by fast EPSPs in many myenteric neurons. The model also predicted that synchronous activation of GABAA receptors and fast EPSPs potentiated firing evoked by the latter, while synchronous activation of GABAC receptors with fast EPSPs, potentiated firing and then suppressed it. The results reveal that so-called slow EPSPs have a biphasic effect being likely to suppress fast EPSP evoked firing over very long periods, perhaps accounting for prolonged quiescent periods seen in enteric motor patterns.

L-Cysteine Alleviates Myenteric Neuron Injury Induced by Intestinal Ischemia/Reperfusion via Inhibitin the Macrophage NLRP3-IL-1β Pathway.

Ischemia/reperfusion injury is a common pathophysiological process in the clinic. It causes various injuries, multiple organ dysfunction, and even death. There are several possible mechanisms about ischemia/reperfusion injury, but the influence on intestinal myenteric neurons and the underlying mechanism are still unclear. C57BL6/J mice were used to establish the ischemia/reperfusion model in vivo. Peritoneal macrophages were used for ATP depletion and hypoxia/reoxygenation experiment in vitro. L-cysteine, as the substrate of hydrogen sulfide, is involved in many physiological and pathological processes, including inflammation, metabolism, neuroprotection, and vasodilation. In the current study, we confirmed that intestinal ischemia/reperfusion led to the injury of myenteric neurons. From experiments in vitro and in vivo, we demonstrated that L-cysteine protected myenteric neurons from the injury. AOAA reversed the protective effect of L-cysteine. Also, L-cysteine played a protective role mainly by acting on intestinal macrophages via decreasing the expression of NLRP3, cleaved caspase-1, and mature IL-1β. L-cysteine increased cystathionine beta synthase and H2S produced by intestinal macrophages to protect myenteric mature neurons and enteric neural precursor cells from apoptosis. Moreover, the addition of IL-1β-neutralizing antibody alleviated the injury of myenteric neurons and enteric neural precursor cells caused by intestinal ischemia/reperfusion. Our study provided a new target for the protection of myenteric neurons in clinical intestinal ischemia/reperfusion injury.

Transduction of Systemically Administered Adeno-Associated Virus in the Colonic Enteric Nervous System and c-Kit Cells of Adult Mice.

Systemic delivery of adeno-associated virus (AAV) vectors transduces the enteric nervous system. However, less is known on the mapping and morphological and neurochemical characterization in the adult mouse colon. We used AAV9-CAG-GFP (AAV9) and AAV-PHP.S-hSyn1-tdTomato farnesylated (PHP.S-tdTf) to investigate the segmental distribution, morphologies and neurochemical coding of the transduction. The vectors were retro-orbitally injected in male and female adult mice, and 3 weeks later, the colon was prepared for microcopy with or without immunohistochemistry for neuronal and non-neuronal markers. In contrast to the distributions in neonatal and juvenile rodents, the AAV transduction in neurons and/or nerve fibers was the highest in the proximal colon, decreased gradually in the transverse, and was sparse in the distal colon without difference between sexes. In the proximal colon, the AAV9-transduced myenteric neurons were unevenly distributed. The majority of enteric neurons did not have AAV9 expression in their processes, except those with big soma with or without variously shaped dendrites, and a long axon. Immunolabeling demonstrated that about 31% neurons were transduced by AAV9, and the transduction was in 50, 28, and 31% of cholinergic, nitrergic, and calbindin-positive myenteric neurons, respectively. The nerve fiber markers, calcitonin gene-related peptide alpha, tyrosine hydroxylase or vasoactive intestinal polypeptide co-localized with AAV9 or PHP.S-tdTf in the mucosa, and rarely in the myenteric plexus. Unexpectedly, AAV9 expression appeared also in a few c-Kit immunoreactive cells among the heavily populated interstitial cells of Cajal (ICC). In the distal colon, the AAV transduction appeared in a few nerve fibers mostly the interganglionic strands. Other types of AAV9 and AAV-PHP vectors induced a similar colonic segmental difference which is not colon specific since neurons were transduced in the small intestine and gastric antrum, while little in the gastric corpus and none in the lower esophagus.ConclusionThese findings demonstrate that in adult mice colon that there is a rostro-caudal decrease in the transduction of systemic delivery of AAV9 and its variants independent of sex. The characterization of AAV transduction in the proximal colon in cholinergic and nitrergic myenteric neurons along with a few ICC suggests implications in circuitries regulating motility.

An inter-organ neural circuit for appetite suppression

Glucagon-like peptide-1 (GLP-1) is a signal peptide released from enteroendocrine cells of the lower intestine. GLP-1 exerts anorectic and antimotility actions that protect the body against nutrient malabsorption. However, little is known about how intestinal GLP-1 affects distant organs despite rapid enzymatic inactivation. We show that intestinal GLP-1 inhibits gastric emptying and eating via intestinofugal neurons, a subclass of myenteric neurons that project to abdominal sympathetic ganglia. Remarkably, cell-specific ablation of intestinofugal neurons eliminated intestinal GLP-1 effects, and their chemical activation functioned as a GLP-1 mimetic. GLP-1 sensing by intestinofugal neurons then engaged a sympatho-gastro-spinal-reticular-hypothalamic pathway that links abnormal stomach distension to craniofacial programs for food rejection. Within this pathway, cell-specific activation of discrete neuronal populations caused systemic GLP-1-like effects. These molecularly identified, delimited enteric circuits may be targeted to ameliorate the abdominal bloating and loss of appetite typical of gastric motility disorders.

Adverse effects of methylene blue in peripheral neurons: An in vitro electrophysiology and cell culture study.

Methylene blue (MB) is an effective treatment for methemoglobinemia, ifosfamide-induced encephalopathy, cyanide poisoning, and refractory vasoplegia. However, clinical case reports and preclinical studies indicate potentially neurotoxic activity of MB at certain concentrations. The exact mechanisms of MB neurotoxicity are not known, and while the effects of MB on neuronal tissue from different brain regions and myenteric ganglia have been examined, its effects on primary afferent neurons from dorsal root ganglia (DRG) have not been studied. Mouse DRG were exposed to MB (0.3-10μM) in vitro to assess neurite outgrowth. Increasing concentrations of MB (0.3-10μM) were associated with neurotoxicity as shown by a substantial loss of cells with neurite formation, particularly at 10μM. In parallel experiments, cultured rat DRG neurons were treated with MB (100μM) to examine how MB affects electrical membrane properties of small-diameter sensory neurons. MB decreased peak inward and outward current densities, decreased action potential amplitude, overshoot, afterhyperpolarization, increased action potential rise time, and decreased action potential firing in response to current stimulation. MB induced dose-dependent toxicity in peripheral neurons, in vitro. These findings are consistent with studies in brain and myenteric ganglion neurons showing increased neuronal loss and altered membrane electrical properties after MB application. Further research is needed to parse out the toxicity profile for MB to minimize damage to neuronal structures and reduce side effects in clinical settings.

Long COVID-19 and achalasia: a possible relationship?

Achalasia is a rare esophageal motor disorder with a worldwide prevalence of around 10 cases per 100 000 inhabitants, and an incidence of one new case per 100 000 inhabitants per year. It is characterized by loss or decrease of myenteric plexus neurons in the distal esophagus and lower esophageal sphincter, presenting dysphagia and regurgitation. The objective of this work was to show that the presence of type II achalasia could be a sequela of the COVID-19 infection. Patient histories were reviewed during the 2015-2021 period, the frequencies of achalasia with and without COVID-19 were calculated. Patient profiles were constructed by using cluster analysis based on clinical variables. It was found that frequency of patients with achalasia during the years 2020 and 2021 was higher than that observed in previous years, and by the year 2021, 2/3 of the patients with achalasia had presented COVID-19 infection, in addition, the patients with type I achalasia presented different profiles than patients with type II achalasia according to the cluster analysis, and the frequency of COVID-19 was much lower in patients with type I achalasia. These results seem to indicate type II achalasia could be a sequela of COVID-19 infection. The possible etiopathogenic implications of these results are discussed, as well as their clinical relevance.

Localization of substance P (SP)-immunoreactivity in the myenteric plexus of the rat esophagus.

The present immunohistochemical study was performed to examine the number, distribution, and chemical coding of intrinsic substance P (SP) neurons and nerve fibers within the esophagus and discuss their functional roles. Many SP neurons and nerve fibers were found in the myenteric plexus, and the SP neurons gradually decreased from the oral side toward the aboral side of the esophagus. Double-immunolabeling showed that most SP neurons were cholinergic (positive for choline acetyltransferase), and few were nitrergic (positive for nitric oxide synthase). Some cholinergic SP nerve terminals surrounded cell bodies of several myenteric neurons. In the muscularis mucosa and lower esophageal sphincter, and around blood vessels, numerous SP nerve endings were present, and many of them were cholinergic. Also, SP nerve endings were found on only a few motor endplates of the striated muscles, and most of them were calcitonin gene-related peptide (CGRP)-positive. Retrograde tracing using Fast Blue (FB) showed that numerous sensory neurons in the dorsal root ganglia (DRGs) and nodose ganglion (NG) projected to the esophagus, and most FB-labeled SP neurons were CGRP-positive. These results suggest that the intrinsic SP neurons in the rat esophagus may play roles as, at least, motor neurons, interneurons, and vasomotor neurons, which are involved in local regulation of smooth muscle motility, neuronal transmission, and blood circulation, respectively. Moreover, SP nerve endings on only a minority of motor endplates may be extrinsic, derived from DRGs or NG, and possibly detect chemical circumstances within motor endplates to modulate esophageal motility.

Topographical Organization and Morphology of Substance‐P Axons in the Flat‐Mounts of the Mouse Whole Stomach

More than 50 million Americans suffer from chronic pain. However, the anatomical and physiological mechanisms of peripheral nociceptive processes have not been well elucidated which has seriously impeded the progress of designing novel bioengineering manipulations/treatments for chronic pain. In this study, we performed a comprehensive topographical mapping of pain‐related neural circuitry in the flat‐mount of whole mouse stomach (male, n=8, 3‐5 months). We used Substance P (SP) as a marker for nociceptive axons and applied a combination of state‐of‐the‐art techniques, including flat‐mount tissue processing and immunohistochemistry of the whole stomach, confocal microscopy, Zeiss Imager microscopy to determine the distribution and morphology of SP‐IR axons and terminals in the whole stomach. We found that 1) SP‐IR axons formed extensive terminal networks in both the ventral and dorsal stomachs. 2) SP‐IR axons were much denser in the antrum and corpus regions than in the fundus and cardia. 3) SP‐IR varicose axons ran in parallel with the circular and longitudinal muscle layers. 4) SP‐IR axons innervated blood vessels. 5) SP‐IR varicose axons formed terminals wrapping around individual myenteric neurons. 6) There were no confirmed SP‐IR myenteric neurons. 7) SP‐IR afferent innervation of the stomach showed a similar pattern of SP‐IR axons and terminals between the ventral and dorsal stomachs. In control mice (n=8), we injected tracer DiI into the ventral and dorsal stomach muscular layers or Fluorogold injection (i.p) and found that the main extrinsic source of SP‐IR afferent axons in the stomach was from the T7‐T11 DRG and to a lesser extent the VNG, but not from the celiac ganglia and dorsal motor nucleus of vagus. Our data provide for the first time a comprehensive topographical map for SP‐IR axons and terminals in the whole stomach with single cell/axon/synapse resolution. This work will contribute to a 3D digital representation of a brain‐stomach nociceptive atlas in a stomach scaffold, thus providing a novel anatomical foundation for functional mapping of nociceptive afferent axons and their pathological remodeling in the stomach. The first two authors contributed equally to this work.