Circuit-specific enteric glia regulate intestinal motor neurocircuits.

Glia in the central nervous system exert precise spatial and temporal regulation over neural circuitry on a synapse-specific basis, but it is unclear if peripheral glia share this exquisite capacity to sense and modulate circuit activity. In the enteric nervous system (ENS), glia control gastrointestinal motility through bidirectional communication with surrounding neurons. We combined glial chemogenetics with genetically encoded calcium indicators expressed in enteric neurons and glia to study network-level activity in the intact myenteric plexus of the proximal colon. Stimulation of neural fiber tracts projecting in aboral, oral, and circumferential directions activated distinct populations of enteric glia. The majority of glia responded to both oral and aboral stimulation and circumferential pathways, while smaller subpopulations were activated only by ascending and descending pathways. Cholinergic signaling functionally specifies glia to the descending circuitry, and this network plays an important role in repressing the activity of descending neural pathways, with some degree of cross-inhibition imposed upon the ascending pathway. Glial recruitment by purinergic signaling functions to enhance activity within ascending circuit pathways and constrain activity within descending networks. Pharmacological manipulation of glial purinergic and cholinergic signaling differentially altered neuronal responses in these circuits in a sex-dependent manner. Collectively, our findings establish that the balance between purinergic and cholinergic signaling may differentially control specific circuit activity through selective signaling between networks of enteric neurons and glia. Thus, enteric glia regulate the ENS circuitry in a network-specific manner, providing profound insights into the functional breadth and versatility of peripheral glia.

Sympathetic Input to Multiple Cell Types in Mouse and Human Colon Produces Region-Specific Responses.

BACKGROUND & AIMS: The colon is innervated by intrinsic and extrinsic neurons that coordinate functions necessary for digestive health. Sympathetic input suppresses colon motility by acting on intrinsic myenteric neurons, but the extent of sympathetic-induced changes on large-scale network activity in myenteric circuits has not been determined. Compounding the complexity of sympathetic function, there is evidence that sympathetic transmitters can regulate activity in non-neuronal cells (such as enteric glia and innate immune cells). METHODS: We performed anatomical tracing, immunohistochemistry, optogenetic (GCaMP calcium imaging, channelrhodopsin), and colon motility studies in mice and single-cell RNA sequencing in human colon to investigate how sympathetic postganglionic neurons modulate colon function. RESULTS: Individual neurons in each sympathetic prevertebral ganglion innervated the proximal or distal colon, with processes closely opposed to multiple cell types. Calcium imaging in semi-intact mouse colon preparations revealed changes in spontaneous and evoked neural activity, as well as activation of non-neuronal cells, induced by sympathetic nerve stimulation. The overall pattern of response to sympathetic stimulation was unique to the proximal or distal colon. Region-specific changes in cellular activity correlated with motility patterns produced by electrical and optogenetic stimulation of sympathetic pathways. Pharmacology experiments (mouse) and RNA sequencing (human) indicated that appropriate receptors were expressed on different cell types to account for the responses to sympathetic stimulation. Regional differences in expression of α-1 adrenoceptors in human colon emphasize the translational relevance of our mouse findings. CONCLUSIONS: Sympathetic neurons differentially regulate activity of neurons and non-neuronal cells in proximal and distal colon to promote distinct changes in motility patterns, likely reflecting the distinct roles played by these 2 regions.

Intestinal Bacteria Maintain Adult Enteric Nervous System and Nitrergic Neurons via Toll-like Receptor 2-induced Neurogenesis in Mice.

BACKGROUND & AIMS: The enteric nervous system (ENS) exists in close proximity to luminal bacteria. Intestinal microbes regulate ENS development, but little is known about their effects on adult enteric neurons. We investigated whether intestinal bacteria or their products affect the adult ENS via toll-like receptors (TLRs) in mice. METHODS: We performed studies with conventional C57/BL6, germ-free C57/BL6, Nestin-creERT2:tdTomato, Nestin-GFP, and ChAT-cre:tdTomato. Mice were given drinking water with ampicillin or without (controls). Germ-free mice were given drinking water with TLR2 agonist or without (controls). Some mice were given a blocking antibody against TLR2 or a TLR4 inhibitor. We performed whole gut transit, bead latency, and geometric center studies. Feces were collected and analyzed by 16S ribosomal RNA gene sequencing. Longitudinal muscle myenteric plexus (LMMP) tissues were collected, analyzed by immunohistochemistry, and levels of nitric oxide were measured. Cells were isolated from colonic LMMP of Nestin-creERT2:tdTomato mice and incubated with agonists of TLR2 (receptor for gram-positive bacteria), TLR4 (receptor for gram-negative bacteria), or distilled water (control) and analyzed by flow cytometry. RESULTS: Stool from mice given ampicillin had altered composition of gut microbiota with reduced abundance of gram-positive bacteria and increased abundance of gram-negative bacteria, compared with mice given only water. Mice given ampicillin had reduced colon motility compared with mice given only water, and their colonic LMMP had reduced numbers of nitrergic neurons, reduced neuronal nitric oxide synthase production, and reduced colonic neurogenesis. Numbers of colonic myenteric neurons increased after mice were switched from ampicillin to plain water, with increased markers of neurogenesis. Nestin-positive enteric neural precursor cells expressed TLR2 and TLR4. In cells isolated from the colonic LMMP, incubation with the TLR2 agonist increased the percentage of neurons originating from enteric neural precursor cells to approximately 10%, compared with approximately 0.01% in cells incubated with the TLR4 agonist or distilled water. Mice given an antibody against TLR2 had prolonged whole gut transit times; their colonic LMMP had reduced total neurons and a smaller proportion of nitrergic neurons per ganglion, and reduced markers of neurogenesis compared with mice given saline. Colonic LMMP of mice given the TLR4 inhibitor did not have reduced markers of neurogenesis. Colonic LMMP of germ-free mice given TLR2 agonist had increased neuronal numbers compared with control germ-free mice. CONCLUSIONS: In the adult mouse colon, TLR2 promotes colonic neurogenesis, regulated by intestinal bacteria. Our findings indicate that colonic microbiota help maintain the adult ENS via a specific signaling pathway. Pharmacologic and probiotic approaches directed towards specific TLR2 signaling processes might be developed for treatment of colonic motility disorders related to use of antibiotics or other factors.

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

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

Estrogen receptor ß controls proliferation of enteric glia and differentiation of neurons in the myenteric plexus after damage.

Injury to the enteric nervous system (ENS) can cause several gastrointestinal (GI) disorders including achalasia, irritable bowel syndrome, and gastroparesis. Recently, a subpopulation of enteric glial cells with neuronal stem/progenitor properties (ENSCs) has been identified in the adult ENS. ENSCs have the ability of reconstituting the enteric neuronal pool after damage of the myenteric plexus. Since the estrogen receptor ß (ERß) is expressed in enteric glial cells and neurons, we investigated whether a selective ERß agonist, LY3201, can influence neuronal and glial cell differentiation. Myenteric ganglia from the murine muscularis externa were isolated and cultured in either glial cell medium or neuronal medium. In glial cell medium, the number of glial progenitor cells (Sox10+) was increased by fourfold in the presence of LY3201. In the neuronal medium supplemented with an antimitotic agent to block glial cell proliferation, LY3201 elicited a 2.7-fold increase in the number of neurons (neurofilament+ or HuC/D+). In addition, the effect of LY3201 was evaluated in vivo in two murine models of enteric neuronal damage and loss, namely, high-fat diet and topical application of the cationic detergent benzalkonium chloride (BAC) on the intestinal serosa, respectively. In both models, treatment with LY3201 significantly increased the recovery of neurons after damage. Thus, LY3201 was able to stimulate glial-to-neuron cell differentiation in vitro and promoted neurogenesis in the damaged myenteric plexus in vivo. Overall, our study suggests that selective ERß agonists may represent a therapeutic tool to treat patients suffering from GI disorders, caused by excessive neuronal/glial cell damage.

Histamine-dependent interactions between mast cells, glia, and neurons are altered following early-life adversity in mice and humans.

Early-life adversity contributes to the development of functional bowel disorders later in life through unresolved mechanisms. Here, we tested the hypothesis that early-life adversity alters anatomical and functional interactions between mast cells and enteric glia. The effects of early-life stress were studied using the neonatal maternal separation (NMS) stress mouse model. Anatomical relationships between mast cells and enteric glia were assessed using immunohistochemistry and mast cell reporter mice (Mcpt5Cre;GCaMP5g-tdT). Immunohistochemistry was used to assess the expression of histamine, histamine 1 (H1) receptors, and glial fibrillary acidic protein. Functional responses of glia to mast cell mediators were assessed in calcium imaging experiments using Sox10CreERT2;GCaMP5g-tdT mice and cultured human enteric glial cells. NMS increases mast cell numbers at the level of the myenteric plexus and their proximity to myenteric ganglia. Myenteric glia respond to mediators released by activated mast cells that are blocked by H1 receptor antagonists in mice and humans and by blocking neuronal activity with tetrodotoxin in mouse tissue. Histamine replicates the effects of mast cell supernatants on enteric glia, and NMS increases histamine production by mast cells. NMS reduces glial responses to mast cell mediators in mouse tissue, while potentiating responses in cultured human enteric glia. NMS increases myenteric glial fibrillary acidic protein expression and reduces glial process length but does not cause neurodegeneration. Histamine receptor expression is not altered by NMS and is localized to neurons in mice, but glia in humans. Early-life stress increases the potential for interactions between enteric glia and mast cells, and histamine is a potential mediator of mast cell-glial interactions through H1 receptors. We propose that glial-mast cell signaling is a mechanism that contributes to enteric neuroplasticity driven by early-life adversity.NEW & NOTEWORTHY Early-life adversity places an individual at risk for developing functional gastrointestinal disorders later in life through unknown mechanisms. Here, we show that interactions between mast cells and glia are disrupted by early-life stress in mice and that histamine is a potential mediator of mast cell-glial interactions.

Extrinsic Primary Afferent Neurons Link Visceral Pain to Colon Motility Through a Spinal Reflex in Mice.

BACKGROUND & AIMS: Proper colon function requires signals from extrinsic primary afferent neurons (ExPANs) located in spinal ganglia. Most ExPANs express the vanilloid receptor TRPV1, and a dense plexus of TRPV1-positive fibers is found around myenteric neurons. Capsaicin, a TRPV1 agonist, can initiate activity in myenteric neurons and produce muscle contraction. ExPANs might therefore form motility-regulating synapses onto myenteric neurons. ExPANs mediate visceral pain, and myenteric neurons mediate colon motility, so we investigated communication between ExPANs and myenteric neurons and the circuits by which ExPANs modulate colon function. METHODS: In live mice and colon tissues that express a transgene encoding the calcium indicator GCaMP, we visualized levels of activity in myenteric neurons during smooth muscle contractions induced by application of capsaicin, direct colon stimulation, stimulation of ExPANs, or stimulation of preganglionic parasympathetic neuron (PPN) axons. To localize central targets of ExPANs, we optogenetically activated TRPV1-expressing ExPANs in live mice and then quantified Fos immunoreactivity to identify activated spinal neurons. RESULTS: Focal electrical stimulation of mouse colon produced phased-locked calcium signals in myenteric neurons and produced colon contractions. Stimulation of the L6 ventral root, which contains PPN axons, also produced myenteric activation and contractions that were comparable to those of direct colon stimulation. Surprisingly, capsaicin application to the isolated L6 dorsal root ganglia, which produced robust calcium signals in neurons throughout the ganglion, did not activate myenteric neurons. Electrical activation of the ganglia, which activated even more neurons than capsaicin, did not produce myenteric activation or contractions unless the spinal cord was intact, indicating that a complete afferent-to-efferent (PPN) circuit was necessary for ExPANs to regulate myenteric neurons. In TRPV1-channel rhodopsin-2 mice, light activation of ExPANs induced a pain-like visceromotor response and expression of Fos in spinal PPN neurons. CONCLUSIONS: In mice, ExPANs regulate myenteric neuron activity and smooth muscle contraction via a parasympathetic spinal circuit, linking sensation and pain to motility.

Interactions Between Commensal Bacteria and Enteric Neurons, via FPR1 Induction of ROS, Increase Gastrointestinal Motility in Mice.

BACKGROUND & AIMS: Reduced gastrointestinal (GI) motility is a feature of disorders associated with intestinal dysbiosis and loss of beneficial microbes. It is not clear how consumption of beneficial commensal microbes, marketed as probiotics, affects the enteric nervous system (ENS). We studied the effects of the widely used probiotic and the commensal Lactobacillus rhamnosus GG (LGG) on ENS and GI motility in mice. METHODS: Conventional and germ free C57B6 mice were gavaged with LGG and intestinal tissues were collected; changes in the enteric neuronal subtypes were assessed by real-time polymerase chain reaction, immunoblots, and immunostaining. Production of reactive oxygen species (ROS) in the jejunal myenteric plexi and phosphorylation (p) of mitogen-activated protein kinase 1 (MAPK1) in the enteric ganglia were assessed by immunoblots and immunostaining. Fluorescence in situ hybridization was performed on jejunal cryosections with probes to detect formyl peptide receptor 1 (FPR1). GI motility in conventional mice was assessed after daily gavage of LGG for 1 week. RESULTS: Feeding of LGG to mice stimulated myenteric production of ROS, increased levels of phosphorylated MAPK1, and increased expression of choline acetyl transferase by neurons (P < .001). These effects were not observed in mice given N-acetyl cysteine (a ROS inhibitor) or LGGΩSpaC (an adhesion-mutant strain of LGG) or FPR1-knockout mice. Gavage of mice with LGG for 1 week significantly increased stool frequency, reduced total GI transit time, and increased contractions of ileal circular muscle strips in ex vivo experiments (P < .05). CONCLUSIONS: Using mouse models, we found that LGG-mediated signaling in the ENS requires bacterial adhesion, redox mechanisms, and FPR1. This pathway might be activated to increase GI motility in patients.

Effect of acetylsalicylic acid on total myenteric neurons in mice experimentally infected with Trypanosoma cruzi.

We investigated the effects of acetylsalicylic acid (ASA) on the total myenteric neuronal population in the descending colon in Trypanosoma cruzi-infected mice. Thirty-five male Swiss mice, 60 days old, were divided into a control group (C group), control group treated with ASA (CA group), infected group (I group), and infected group treated with ASA (IA group). A total of 1300 trypomastigotes of the Y strain of T. cruzi were intraperitoneally inoculated in the IA and I groups. The CA and IA groups were treated with ASA intraperitoneally. At 75 days post-infection (dpi), all of the animals were sacrificed. Neurons in the colon were stained with Giemsa, quantified, and measured. No difference in the course of infection was observed between the IA and I groups, reflected by the parasitemia curve. Acetylsalicylic acid treatment in the CA and IA groups did not alter the total number of myenteric neurons compared with the C and I groups. The CA and IA groups exhibited an increase in the nuclear area, cytoplasmic area, and neuronal body area compared with the C and I groups. Future studies should elucidate the mechanism of action of ASA against Chagas' disease in the chronic phase.

Expression of P2X1 receptors in somatostatin-containing cells in mouse gastrointestinal tract and pancreatic islets of both mouse and human.

With immunohistochemical and Western blot techniques, P2X1 receptors were detected in the whole mouse gastrointestinal tract and pancreatic islets of mouse and human. (1) δ Cells containing somatostatin (SOM) in the stomach corpus, small intestines, distal colon, pancreatic islets of both mouse and human express P2X1 receptors; (2) strong immunofluorescence of P2X1 receptors was detected in smooth muscle fibers and capillary networks of the villus core of mouse intestine; and (3) P2X1 receptor-immunoreactive neurons were also detected widely in both mouse myenteric and submucosal plexuses, all of which express SOM. The present data implies that ATP via P2X1 receptors is involved in SOM release from pancreatic δ cells, enteric neurons, and capillary networks in villi.

Neuroprotective effects of vitamin D on high fat diet- and palmitic acid-induced enteric neuronal loss in mice.

BACKGROUND: The role of vitamin D in obesity and diabetes is debated. Obese and/or diabetic patients have elevated levels of free fatty acids, increased susceptibility to gastrointestinal symptoms and are suggested to have altered vitamin D balance. The enteric nervous system is pivotal in regulating gastrointestinal activity and high fat diet (HFD) has been shown to cause loss of enteric neurons in ileum and colon. This study investigates the effect of vitamin D on HFD- and palmitic acid-induced enteric neuronal loss in vivo and in vitro. METHODS: Mice were fed either a normal diet (ND) or HFD supplemented with varying levels of vitamin D (from 0x to 20x normal vitamin D level) for 19 weeks. Ileum and colon were analyzed for neuronal numbers and remodeling. Primary cultures of myenteric neurons from mouse small intestine were treated with palmitic acid (4x10-4M) and/or 1α,25-hydroxy-vitamin D3 (VD, 10-11- 10-7M) with or without modulators of lipid metabolism and VD pathways. Cultures were analyzed by immunocyto- and histochemical methods. RESULTS: Vitamin D supplementation had no effect on enteric neuronal survival in the ND group. HFD caused substantial loss of myenteric neurons in ileum and colon. Vitamin D supplementation between 0-2x normal had no effect on HFD-induced neuronal loss. Supplementation with 20x normal, prevented the HFD-induced neuronal loss. In vitro supplementation of VD prevented the palmitic acid-induced neuronal loss. The VD receptor (VDR) was not identified in enteric neurons. Enteric glia expressed the alternative VD receptor, protein disulphide isomerase family A member 3 (PDIA3), but PDIA3 was not found to mediate the VD response in vitro. Inhibition of peroxisome proliferator-activated receptor gamma (PPARγ) and immune neutralization of isocitrate lyase prevented the VD mediated neuroprotection to palmitic acid exposure. CONCLUSIONS: Results show that VD protect enteric neurons against HFD and palmitic acid induced neuronal loss. The mechanism behind is suggested to be through activation of PPARγ leading to improved neuronal peroxisome function and metabolism of neuronal lipid intermediates.

Assemblages A and B of Giardia duodenalis reduce enteric glial cells in the small intestine in mice.

Infection of Giardia duodenalis is one of the most common human parasitic disease worldwide. This infection may be related to important changes in the enteric nervous system. The objective of this study was to evaluate the myenteric and submucosal plexuses, the intestinal muscle layer, and gastrointestinal transit in mice infected with assemblages A and B of G. duodenalis. Swiss albino mice (Mus musculus) were infected with assemblages A and B of G. duodenalis for 15 days. Gastrointestinal transit time was evaluated before euthanasia. Duodenum and jejunum were removed for histological and immunohistochemical analyses. It was observed a reduction in the enteric glial cell count and a decrease in the ratio of enteric glial cells to neurons. The number of neurons did not change, but morphological changes were observed in the duodenum and jejunum in both plexuses, including an increase in the nuclear area and a reduction of cell bodies in the myenteric plexus and a decrease in the nuclear area in the submucosal plexus. A reduction of the thickness of the muscle layer was observed in the duodenum, with no significant differences in the gastrointestinal transit times. Assemblages A and B of G. duodenalis decrease the number of enteric glial cells in the myenteric and submucosal plexuses, decrease the thickness of the muscle layer, and change the morphology of neurons. Graphical abstract ᅟ.

Calbindin D28k-Immunoreactivity in Human Enteric Neurons.

Calbindin (CALB) is well established as immunohistochemical marker for intrinsic primary afferent neurons in the guinea pig gut. Its expression by numerous human enteric neurons has been demonstrated but little is known about particular types of neurons immunoreactive for CALB. Here we investigated small and large intestinal wholemount sets of 26 tumor patients in order to evaluate (1) the proportion of CALB⁺ neurons in the total neuron population, (2) the colocalization of CALB with calretinin (CALR), somatostatin (SOM) and vasoactive intestinal peptide (VIP) and (3) the morphology of CALB+ neurons. CALB+ neurons represented a minority of myenteric neurons (small intestine: 31%; large intestine: 25%) and the majority of submucosal neurons (between 72 and 95%). In the submucosa, most CALB⁺ neurons co-stained for CALR and VIP (between 69 and 80%) or for SOM (between 20 and 3%). In the myenteric plexus, 85% of CALB+ neurons did not co-stain with the other markers investigated. An unequivocal correlation between CALB reactivity and neuronal morphology was found for myenteric type III neurons in the small intestine: uniaxonal neurons with long, slender and branched dendrites were generally positive for CALB. Since also other neurons displayed occasional CALB reactivity, this protein is not suited as an exclusive marker for type III neurons.

Mechanism of Activation of Enteric Nociceptive Neurons via Interaction of TLR4 and TRPV1 Receptors.

Evidence obtained by immunohistochemical double labeling and confocal laser scanning microscopy suggests that capsaicin, a ligand of the TRPV1 nociceptive vanilloid receptor, increases the number of TLR4-positive neurons in the rat colon myenteric plexus. In colitis caused by trinitrobenzene sulfonate, an increase in TRPV1 expression was more significant in both plexuses. Specific inhibitor of the TLR4 (C34) pattern-recognition receptor reduces TRPV1 expression in enteric neurons of both intact rats and rats with induced acute colitis. Thus, stimulation of nociceptive neurons by means of direct activation of their receptors of innate immunity (TLR4) is one of the possible mechanisms underlying the visceral pain in bacterial invasion and inflammatory bowel diseases.

The acute inhibition of enteric glial metabolism with fluoroacetate alters calcium signaling, hemichannel function, and the expression of key proteins.

Glia play key roles in the regulation of neurotransmission in the nervous system. Fluoroacetate (FA) is a metabolic poison widely used to study glial functions by disrupting the tricarboxylic acid cycle enzyme aconitase. Despite the widespread use of FA, the effects of FA on essential glial functions such as calcium (Ca2+) signaling and hemichannel function remain unknown. Therefore, our goal was to assess specifically the impact of FA on essential glial cell functions that are involved with neurotransmission in the enteric nervous system. To this end, we generated a new optogenetic mouse model to study specifically the effects of FA on enteric glial Ca2+ signaling by crossing PC::G5-tdTomato mice with Sox10::creERT2 mice. FA did not change the peak glial Ca2+ response when averaged across all glia within a ganglion. However, FA decreased the percent of responding glia by 30% (P < 0.05) and increased the peak Ca2+ response of the glial cells that still exhibited a response by 26% (P < 0.01). Disruption of Ca2+ signaling with FA impaired the activity-dependent uptake of ethidium bromide through connexin-43 (Cx43) hemichannels (P < 0.05) but did not affect baseline Cx43-dependent dye uptake. FA did not cause overt glial or neurodegeneration, but glial cells significantly increased glial fibrillary acid protein by 56% (P < 0.05) following treatment with FA. Together, these data show that the acute impairment of glial metabolism with FA causes key changes in glial functions associated with their roles in neurotransmission and phenotypic changes indicative of reactive gliosis. NEW & NOTEWORTHY: Our study shows that the acute impairment of enteric glial metabolism with fluoroacetate (FA) alters specific glial functions that are associated with the modification of neurotransmission in the gut. These include subtle changes to glial agonist-evoked calcium signaling, the subsequent disruption of connexin-43 hemichannels, and changes in protein expression that are consistent with a transition to reactive glia. These changes in glial function offer a mechanistic explanation for the effects of FA on peripheral neuronal networks.

Homer1 (VesL-1) in the rat esophagus: focus on myenteric plexus and neuromuscular junction.

Homer1, a scaffolding protein of the postsynaptic density (PSD), enriched at excitatory synapses is known to anchor and modulate group I metabotropic glutamate receptors (mGluRs) and different channel- and receptor-proteins. Homer proteins are expressed in neurons of different brain regions, but also in non-neuronal tissues like skeletal muscle. Occurrence and location of Homer1 and mGluR5 in myenteric plexus and neuromuscular junctions (NMJ) of rat esophagus have yet not been characterized. We located Homer1 and mGluR5 immunoreactivity (-iry) in rat esophagus and focused on myenteric neurons, intraganglionic laminar endings (IGLEs) and NMJs, using double- and triple-label immunohistochemistry and confocal laser scanning microscopy. Homer1-iry was found in a subpopulation of vesicular glutamate transporter 2 (VGLUT2) positive IGLEs and cholinergic varicosities within myenteric ganglia, but neither in nitrergic nor cholinergic myenteric neuronal cell bodies. Homer1-iry was detected in 63% of esophageal and, for comparison, in 35% of sternomastoid NMJs. Besides the location in the PSD, Homer1-iry colocalized with cholinergic markers, indicating a presynaptic location in coarse VAChT/CGRP/NF200- immunoreactive (-ir) terminals of nucleus ambiguus neurons supplying striated esophageal muscle. mGluR5-iry was found in subpopulations of myenteric neuronal cell bodies, VGLUT2-ir IGLEs and cholinergic varicosities within the myenteric neuropil and NMJs of esophagus and sternomastoid muscles. Thus, Homer1 may anchor mGluR5 at presynaptic sites of cholinergic boutons at esophageal motor endplates, in a small subpopulation of VGLUT2-ir IGLEs and cholinergic varicosities within myenteric ganglia possibly modulating Ca2+-currents and neurotransmitter release.

Role of muscarinic-3 receptor antibody in systemic sclerosis: correlation with disease duration and effects of IVIG.

Gastrointestinal dysmotility in systemic sclerosis (SSc) is associated with autoantibodies against muscarinic-3 receptor (M3-R). We investigated the temporal course of the site of action of these autoantibodies at the myenteric neurons (MN) vs. the smooth muscle (SM) M3-R in relation to disease duration, and determined the role of intravenous immunoglobulin (IVIG) in reversing these changes. Immunoglobulins purified from SSc patients (SScIgG) were used to assess their differential binding to MN and SM (from rat colon) employing immunohistochemistry (IHC). Effect of SScIgG on neural and direct muscle contraction was determined by cholinergic nerve stimulation and bethanechol-induced SM contraction. Effects of IVIG and its antigen-binding fragment F(ab')2 on SScIgG binding were studied by enzyme-linked immunosorbent assay (ELISA) of rat colonic longitudinal SM myenteric plexus (LSMMP) lysate and to second extracellular loop peptide of M3-R (M3-RL2). SScIgG from all patients demonstrated significantly higher binding to MN than to SM. With progression of SSc duration, binding at MN and SM increased in a linear fashion with a correlation coefficient of 0.696 and 0.726, respectively (P < 0.05). SScIgG-mediated attenuation of neural and direct SM contraction also increased with disease duration. ELISA analysis revealed that IVIG and F(ab')2 significantly reduced SScIgG binding to LSMMP lysate and M3-RL2. Dysmotility in SSc occurs sequentially, beginning with SScIgG-induced blockage of cholinergic neurotransmission (neuropathy), which progresses to inhibition of acetylcholine action at the SM cell (myopathy). IVIG reverses this cholinergic dysfunction at the neural and myogenic receptors by anti-idiotypic neutralization of SScIgG.

Postnatal development of the myenteric glial network and its modulation by butyrate.

The postnatal period is crucial for the development of gastrointestinal (GI) functions. The enteric nervous system is a key regulator of GI functions, and increasing evidences indicate that 1) postnatal maturation of enteric neurons affect the development of GI functions, and 2) microbiota-derived short-chain fatty acids can be involved in this maturation. Although enteric glial cells (EGC) are central regulators of GI functions, the postnatal evolution of their phenotype remains poorly defined. We thus characterized the postnatal evolution of EGC phenotype in the colon of rat pups and studied the effect of short-chain fatty acids on their maturation. We showed an increased expression of the glial markers GFAP and S100ß during the first postnatal week. As demonstrated by immunohistochemistry, a structured myenteric glial network was observed at 36 days in the rat colons. Butyrate inhibited EGC proliferation in vivo and in vitro but had no effect on glial marker expression. These results indicate that the EGC myenteric network continues to develop after birth, and luminal factors such as butyrate endogenously produced in the colon may affect this development.

Inhibiting Inducible Nitric Oxide Synthase in Enteric Glia Restores Electrogenic Ion Transport in Mice With Colitis.

BACKGROUND & AIMS: Disturbances in the control of ion transport lead to epithelial barrier dysfunction in patients with colitis. Enteric glia regulate intestinal barrier function and colonic ion transport. However, it is not clear whether enteric glia are involved in epithelial hyporesponsiveness. We investigated enteric glial regulation of ion transport in mice with trinitrobenzene sulfonic acid- or dextran sodium sulfate-induced colitis and in Il10(-/-) mice. METHODS: Electrically evoked ion transport was measured in full-thickness segments of colon from CD1 and Il10(-/-) mice with or without colitis in Ussing chambers. Nitric oxide (NO) production was assessed using amperometry. Bacterial translocation was investigated in the liver, spleen, and blood of mice. RESULTS: Electrical stimulation of the colon evoked a tetrodotoxin-sensitive chloride secretion. In mice with colitis, ion transport almost completely disappeared. Inhibiting inducible NO synthase (NOS2), but not neuronal NOS (NOS1), partially restored the evoked secretory response. Blocking glial function with fluoroacetate, which is not a NOS2 inhibitor, also partially restored ion transport. Combined NOS2 inhibition and fluoroacetate administration fully restored secretion. Epithelial responsiveness to vasoactive intestinal peptide was increased after enteric glial function was blocked in mice with colitis. In colons of mice without colitis, NO was produced in the myenteric plexus almost completely via NOS1. NO production was increased in mice with colitis, compared with mice without colitis; a substantial proportion of NOS2 was blocked by fluoroacetate administration. Inhibition of enteric glial function in vivo reduced the severity of trinitrobenzene sulfonic acid-induced colitis and associated bacterial translocation. CONCLUSIONS: Increased production of NOS2 in enteric glia contributes to the dysregulation of intestinal ion transport in mice with colitis. Blocking enteric glial function in these mice restores epithelial barrier function and reduces bacterial translocation.

LRIG1 Regulates Ontogeny of Smooth Muscle-Derived Subsets of Interstitial Cells of Cajal in Mice.

BACKGROUND & AIMS: Interstitial cells of Cajal (ICC) control intestinal smooth muscle contraction to regulate gut motility. ICC within the plane of the myenteric plexus (ICC-MY) arise from KIT-positive progenitor cells during mouse embryogenesis. However, little is known about the ontogeny of ICC associated with the deep muscular plexus (ICC-DMP) in the small intestine and ICC associated with the submucosal plexus (ICC-SMP) in the colon. Leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1) marks intestinal epithelial stem cells, but the role of LRIG1 in nonepithelial intestinal cells has not been identified. We sought to determine the ontogeny of ICC-DMP and ICC-SMP, and whether LRIG1 has a role in their development. METHODS: Lrig1-null mice (homozygous Lrig1-CreERT2) and wild-type mice were analyzed by immunofluorescence and transit assays. Transit was evaluated by passage of orally administered rhodamine B-conjugated dextran. Lrig1-CreERT2 mice or mice with CreERT2 under control of an inducible smooth muscle promoter (Myh11-CreERT2) were crossed with Rosa26-LSL-YFP mice for lineage tracing analysis. RESULTS: In immunofluorescence assays, ICC-DMP and ICC-SMP were found to express LRIG1. Based on lineage tracing, ICC-DMP and ICC-SMP each arose from LRIG1-positive smooth muscle progenitors. In Lrig1-null mice, there was loss of staining for KIT in DMP and SMP regions, as well as for 2 additional ICC markers (anoctamin-1 and neurokinin 1 receptor). Lrig1-null mice had significant delays in small intestinal transit compared with control mice. CONCLUSIONS: LRIG1 regulates the postnatal development of ICC-DMP and ICC-SMP from smooth muscle progenitors in mice. Slowed small intestinal transit observed in Lrig1-null mice may be due, at least in part, to loss of the ICC-DMP population.