BACKGROUND AND AIMS: The gastrointestinal (GI) tract extracts nutrients from ingested meals while protecting the organism from infectious agents frequently present in meals. Consequently, most animals conduct the entire digestive process within the GI tract while keeping the luminal contents entirely outside the body, separated by the tightly sealed GI epithelium. Therefore, like the skin and oral cavity, the GI tract must sense the chemical and physical properties of the its external interface to optimize its function. Specialized sensory enteroendocrine cells (EECs) in GI epithelium interact intimately with luminal contents. A subpopulation of EECs express the mechanically gated ion channel Piezo2 and are developmentally and functionally like the skin's touch sensor- the Merkel cell. We hypothesized that Piezo2+ EECs endow the gut with intrinsic tactile sensitivity. METHODS: We generated transgenic mouse models with optogenetic activators in EECs and Piezo2 conditional knockouts. We used a range of reference standard and novel techniques from single cells to living animals, including single-cell RNA sequencing and optoelectrophysiology, opto-organ baths with luminal shear forces, and in vivo studies that assayed GI transit while manipulating the physical properties of luminal contents. RESULTS: Piezo2+ EECs have transcriptomic features of synaptically connected, mechanosensory epithelial cells. EEC activation by optogenetics and forces led to Piezo2-dependent alterations in colonic propagating contractions driven by intrinsic circuitry, with Piezo2+ EECs detecting the small luminal forces and physical properties of the luminal contents to regulate transit times in the small and large bowel. CONCLUSIONS: The GI tract has intrinsic tactile sensitivity that depends on Piezo2+ EECs and allows it to detect luminal forces and physical properties of luminal contents to modulate physiology.

The enteric nervous system consists of more than a dozen types of neurons aggregated into networks of ganglia throughout the gastrointestinal tract, which regulate contractile activity, mucosal secretion, absorption, and local blood flow.1Furness J.B. J Auton Nerv Syst. 2000; 81: 87-96Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar, 2Furness J.B. Nat Rev Gastroenterol Hepatol. 2012; 9: 286-294Crossref PubMed Scopus (866) Google Scholar Mechanisms that contribute to remodeling of the enteric neuronal networks are of great interest. In the central nervous system, it has been suggested that microglia contribute to the fate, connectivity, and identity of neurons during development.3Tremblay M.E. et al.J Neurosci. 2011; 31: 16064-16069Crossref PubMed Scopus (710) Google Scholar Muscularis propria macrophages (MPM) within the enteric nervous system may have similar functions to microglia. Mice homozygous for the osteopetrosis mutation (Csf1op/op) which do not have MPM, have more neurons in the small intestine4Muller P.A. et al.Cell. 2014; 158: 300-313Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar and a higher proportion of gastric neurons that express nitric oxide synthase (NOS1).5Cipriani G. et al.Gastroenterology. 2018; 154: 2122-2136 e12Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar Myenteric neurons serve diverse functions that can be indicated by their morphology, projections and the expression of marker proteins that define their "chemical code." This study finds a previously unidentified role for MPM in altering the chemical code of myenteric neurons. Csf1op/op mice were maintained on a specialized liquid diet to keep their weight comparable with age-matched-wild type (WT) mice (Supplementary Figure 1A). In the myenteric plexus of WT mice, populations of MPM, absent in Csf1op/op mice5Cipriani G. et al.Gastroenterology. 2018; 154: 2122-2136 e12Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar (Supplementary Figure 1B and C, Supplementary Movie 1 and 2), were associated closely with neurons, suggesting functional interactions.6Gabanyi I. et al.Cell. 2016; 164: 378-391Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar We first tested whether the number of choline acetyltransferase+ (ChAT+) neurons was affected by the absence of MPM in Csf1op/op mice (Supplementary Table 1). The density of neurons, defined by Embryonic lethal, abnormal vision, Drosophila-like protein 3/4 (HuC/D) immunoreactivity, was similar between gastric regions in both WT and Csf1op/op mice (Figure 1A–C, Supplementary Figure 2A) (Mann–Whitney test, P = NS; N = 4), yet was higher in Csf1op/op mice than in WT mice (Figure 1D and E) (P < .01, Mann–Whitney test, n = 36 fields, N = 4). Likewise, the density of ChAT+ neurons was higher in Csf1op/op mice compared with WT mice (Figure 1D and E) (P < .001, Mann–Whitney test, n = 36 fields, N = 4). However, in contrast to an increase in the percentage of NOS1+ neurons,5Cipriani G. et al.Gastroenterology. 2018; 154: 2122-2136 e12Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar the percentage of ChAT+ neurons did not differ between Csf1op/op and WT mice (Figure 1D and E) (Mann–Whitney test, n = 36 fields, N = 4). This result suggests that the presence of macrophages alters the proportion of nitrergic but not cholinergic gastric myenteric neurons. Interestingly, in Csf1op/op mice, the combined percentages of NOS1+ (30%) and ChAT+ neurons (72%) exceeded 100% (Supplementary Figure 2B), indicating partial overlap between these markers. Therefore, we investigated whether the number of NOS1+ChAT+ double-labeled neurons was changed in Csf1op/op mice. In Csf1op/op mice, Nitric Oxide Synthase 1 (NOS1+) ChAT+ neurons were more numerous than in WT mice (Figure 2A and B) (Csf1op/op: 7.8 ± 7.1 cells/field; WT, 1.7 ± 1.6 cells/field; 1-way analysis of variance; P < .001; n = 24; N = 4). This result suggests the ability of macrophages to not only modulate the neuronal number but also affect myenteric neuron differentiation. Enteric neurons are not required for bowel colonization by macrophages,7Avetisyan M. et al.Proc Natl Acad Sci U S A. 2018; 115: 4696-4701Crossref PubMed Scopus (48) Google Scholar but macrophages interact with neurons after birth, by expressing genes, such as bone morphogenetic protein 2 (BMP2), needed for macrophage-enteric neuron interaction and neuronal development.4Muller P.A. et al.Cell. 2014; 158: 300-313Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar To test the intrinsic ability of resident macrophages to modify the neuronal chemical code by establishing functional interaction with neurons, we treated Csf1op/op with CSF1 (Colony Stimulating Factor 1) for 7 weeks to populate the stomach with macrophages (Figure 2C). In CSF1-treated Csf1op/op mice, the proportion of NOS1+ChAT+ neurons remained similar to the proportion of NOS1+ChAT+ neurons in Csf1op/op mice (Figure 2A–C) (1-way analysis of variance; n = 24; N = 4). We previously showed that repopulating macrophages in CSF1-treated Csf1op/op mice had a different phenotype from resident macrophages.5Cipriani G. et al.Gastroenterology. 2018; 154: 2122-2136 e12Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar Consistent with this observation, BMP2 was not expressed by macrophages isolated from CSF1-treated Csf1op/op mice (Antibodies and PCR primers listed in Supplementary Tables 2 and 3), whereas BMP2 was expressed by macrophages isolated from WT mice (Figure 2D and E) (Mann–Whitney test; P < .001; N = 4), as reported elsewhere.4Muller P.A. et al.Cell. 2014; 158: 300-313Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar During development, the chemical code of myenteric neurons changes and the overlap between NOS1 and ChAT decreases as neurons mature.8Hao M.M. et al.J Comp Neurol. 2013; 521: 3358-3370Crossref PubMed Scopus (35) Google Scholar Therefore, increased numbers of double-labeled myenteric neurons may reflect incomplete maturation of myenteric neurons in Csf1op/op mice. MPMs functionally interact with enteric neurons starting at 2 weeks of age,7Avetisyan M. et al.Proc Natl Acad Sci U S A. 2018; 115: 4696-4701Crossref PubMed Scopus (48) Google Scholar therefore the role of resident MPM in promoting myenteric neuron maturation likely happens early in life. Interestingly, MPMs that populate the gastric muscularis propria did not express BMP2, a cytokine important for establishing functional interactions between MPMs and neurons during development. Therefore, as previously suggested,4Muller P.A. et al.Cell. 2014; 158: 300-313Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 9Anitha M. et al.Am J Physiol Gastrointest Liver Physiol. 2010; 298: G375-G383Crossref PubMed Scopus (29) Google Scholar BMP2 may be required for the changes in NOS1 and ChAT expression associated with neuronal maturation. Taken together, our results show a role for MPM in enteric neuronal maturation as indicated by the changes in chemical code in gastric myenteric neurons. The mechanisms by which MPM regulate neuronal numbers and chemical codes needs further investigation because it may be significant to the development or plasticity of the adult enteric nervous system and normal gastric function. The authors thank Mrs Kristy Zodrow for her excellent assistance with this work; the Mayo Microscopy and Cell Analysis Core for assistance with the flow cytometry experiment; and Dr Vanda Lennon (Mayo Clinic) for supplying the HuC/D antibody used for the immunohistochemistry study. These studies were approved by the Mayo Clinic Institutional Animal Care and Use Committee. Mice were humanely killed by carbon dioxide exposure followed by cervical dislocation. Mice homozygous for the Csf1op mutation and WT littermates were studied. These mice were bred in-house from a Csf1op/+ colony of hemizygous breeders with founders originating from The Jackson Laboratory (Bar Harbor, ME). Wild-type Csf1+/+ mice were identified by genotyping as previously described.1Furness J.B. J Auton Nerv Syst. 2000; 81: 87-96Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar Csf1op/op mice were maintained on a specialized wet diet (Bio-serv, Frenchtown, NJ) after weaning at 3–4 weeks of age to keep their weight comparable with age-matched WT mice (Supplementary Figure 1A). After 12 weeks of age, Csf1op/op mice were treated with CSF1 (2.5 μg intraperitoneally once daily, recombinant mouse macrophage colony stimulating factor-1 (rmM-CSF); Peprotech, Rocky Hill, NJ) (Figure 2A). The mucosa was removed and muscularis propria was fixed with 4% paraformaldehyde in 0.1 mol/L phosphate buffer for 4 hours. Then, whole mounts were rinsed in 0.1 mol/L phosphate-buffered saline and blocked in the presence of 10% normal donkey serum in phosphate-buffered saline and 0.3% Triton X-100 (Thermo Fisher, Waltham, MA) overnight at 4°C and gastric muscularis propria was labeled with primary antibodies overnight at 4°C. After washing, the tissue was incubated with secondary antibodies (Jackson ImmunoResearch, West Grove, PA), washed, and then incubated with 4',6-diamidino-2-phenylindole dilactate (Invitrogen, Carlsbad, CA) for 30 minutes. Neurons were identified by HuC/D-immunoreactivity (ANNA1, a gift from Dr Vanda Lennon, Mayo Clinic, Rochester, MN), cholinergic neurons using a goat anti-ChAT antibody (EMD Millipore, Burlington, MA), and nitrergic neurons using a rabbit anti-NOS1 antibody (EMD Millipore). Muscularis macrophages were identified using the MHCII primary antibody (eBioscience, Waltham, MA). Controls omitting the primary antibody and controls in double-labeling experiments that used the wrong secondary antibody were performed for all experiments. For quantification, 3 different fields were taken from the corpus and 3 from the antrum. The list of antibodies is shown in Supplementary Table 1. A laser scanning confocal microscope using a 20×, numerical aperture, (NA), 0.95 XLUMPlanFl objective (Olympus, Tokyo, Japan) in Fluoview (Olympus), with the optimal confocal aperture to provide a resolution of 0.994 × 0.994 × 1.13 μm (X × Y × Z), was used. Stacks of confocal images of the entire muscularis propria were collected from 4 different mice (n = 4). For quantification of the labeling, all of the confocal image stacks were flattened into projections using the FV10-ASW Viewer (Olympus). The flattened images were renumbered in random order and the enteric neuronal number was determined while blinded to the source. All cells were counted from fields with dimensions of 636 × 636 μm. Images used for reconstruction and orthogonal view were taken from a Zeiss LSM 780 microscope using either a 40× 1.2 NA water immersion objective at a resolution of 0.415 × 0.415 × 0.444, or a 100 × 1.4 NA oil immersion objective at a resolution of 0.133 × 0.133 × 0.373 μm per pixel. Images were analyzed using Imaris-Microscopy Image Software by Bitplane (Supplementary Figure 1A). Cell sorting was performed using a fluorescence activated cell sorting Aria Cell Sorter cytometer running fluorescence activated cell sorting Diva 6 software (Becton Dickinson, San Jose, CA), located in the Mayo Clinic Flow Cytometry Core Facility. Aliquots of cells were either unstained or stained with individual fluorescently labeled antibodies (Zurich, Switzerland, Supplementary Table 2) to establish instrument voltages, compensation, and appropriate gates. Each positive control tube was initially run without storing the data to ensure that the positive signals were on scale. Data were analyzed using FlowJo X software (Tree Star, Inc, Ashland, OR). Gastric CD45+CD11b+F4/80+ cells were isolated directly into the lysing buffer provided by the RNeasy micro plus kit (Qiagen, Hilden, Germany). The extraction was performed following the instructions provided and the RNA concentration was determined by using a NanoDrop spectrophotometer. The RNA extracted was used for a real-time quantitative reverse-transcription polymerase chain reaction. The SuperScript VILO complementary DNA Synthesis Kit (Invitrogen) was used to generate complementary DNA. Quantitative reverse-transcription polymerase chain reaction was performed on complementary DNA using commercial primer sets (Supplementary Table 3) and RT2SYBR Green/ROX quantitative reverse-transcription polymerase chain reaction master mix according to the manufacturer's instructions (SABiosciences, Frederick, MD). The data were normalized to the expression of the glyceraldehyde-3-phosphate dehydrogenase by transforming the difference in threshold cycle for the gene of interest and the housekeeping gene to the second power, and expressed as the means ± SEM. Data are expressed as scatter plots with medians and quartiles and analyzed by the Mann–Whitney test. A P value less than .05 was considered significant. The method used for statistical analysis of 3 different groups was 1-way analysis of variance with multiple comparisons. Normality was addressed by applying D'Agostino and Pearson normality tests. Statistical analysis was performed with GraphPad Prism (GraphPad Software, La Jolla, CA).Supplementary Figure 2(A) Quantification of the HuC/D+ myenteric neurons in the gastric corpus and antrum of WT and Csf1op/op mice. (B) Percentage of myenteric neurons identified in Csf1op/op and WT mice. Table shows numbers per field and proportions of different types of myenteric neurons in Csf1op/op and WT mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Table 1Sources of Commercial Antibodies Used in Immunohistochemistry ExperimentsSupplierFinal titerHostClonalityCatalog numberResearch resource initiative identifierPrimary antibody Embryonic lethal, abnormal vision, Drosophila-like protein 3/4Gift from Dr V. Lennon (Mayo Clinic)1:500HumanAB_2314657 NOS1Millipore0.33 μg/mLRabbitPolyclonalAB5380AB_91824 ChATMillipore1:100GoatPolyclonalAB144PAB_2079751 F4/80 direct conjugateThermo Fisher0.4 μg/mLRatPolyclonalMF 48020AB_10376287 Major Histocompatibility Complex IIeBioscience1.0 μg/mLRatMonoclonal14-5321-81AB_467560 Protein Gene Product 9.5Thermo Fisher1:400RabbitPolyclonal38-1000AB_2533355Secondary antibody Cy3 anti-goatJackson ImmunoResearch1.75 μg/mLDonkeyPolyclonal705-165-147AB_2307351 Alexa Fluor–488 anti-ratJackson ImmunoResearch2.33 μg/mLDonkeyPolyclonal712-545-150AB_2340683 Cy3 anti-rabbitJackson ImmunoResearch1.75 μg/mLDonkeyPolyclonal711-165-152AB_2307443 Cy5 anti-humanJackson ImmunoResearch1.75 μg/mLDonkeyPolyclonal709-175-149AB_2340539 Open table in a new tab Supplementary Table 2List of Antibodies Used for Sorting Experiments and List of Primers Used for Quantitative Reverse-Transcription Polymerase Chain ReactionAntibodyFluorophoreCatalog numberCompanyF4/80 monoclonal antibody (BM8)Phycoerythrin--cyanine 515-4801-82eBioscenceAnti-mouse CD11bAlexa Fluor 48853-0112-82eBioscenceAnti-mouse CD45Alexa Fluor 45048-0451-82eBioscenceRat IgG2b K isotype controlAPC17-4031-81eBioscenceRat IgG2a K isotype controlPE-cyanine 725-4321-81eBioscence Open table in a new tab Supplementary Table 3List of Primers used for RT-PCRGene symbolUnigene titleForwardReverseBMP2Bone morphogenetic protein 2GGTGATGGCTTCCTTGTACCAGTGAGGCCCATACCAGAAGGapdhGlyceraldehyde-3-phosphate dehydrogenaseQiagenQiagen Open table in a new tab eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJmMmRiYjgyNWU2MWZkYTNjNWJlYjhhMzA2Y2JjMjBhNSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc5MjgzMzQxfQ.mjaf3jL887okh9sLbhYS7R9nsdGQuw9e5Ju6zJQ4JBXPXEJKKc5jokj_E9Kk94kXZjX6oYaV3UAvvh6k8hMhYw37-2U3nKVFESghXJ76d4PwxTfjJpCZ3nMB4JQgmGeBsD4VulHQmHevBluB89ghQhcdpSePyhrRxS70Q4JfYX_lj35LItc-P-7lfEQlODXaISl5lp8NPWPwZFxHDrTP5bAJOfKI2NaNGUcuGkri10IB68hH90_urJ2H9O5oCNz0LeIGGgaJjfaczIfV-i5XK2_pFaWy1xEwjj1lJKPAFAmNcBJ39G4XHFPoqwaW2IppZ1x_MNbJ1_o52_eJKPaDkg Download .mp4 (1.88 MB) Help with .mp4 files Supplementary Movie 1Macrophage (green)- and nerve fiber (red) distribution in the gastric muscularis propria.eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJhYmNlNmZjMTY2ZGI0NjIyZjY5ZjU4NGVlZmI2YTY3NyIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc5MjgzMzQxfQ.JX0NbPBxxWNUWPlBOLHyUtVj9cYzGu8rw-3XcWLvzJ5WVGw0g5GHDfHMxZ-l3tQjMtklx7Kt8AzfdAbAouCHXE-yF27f6SdltG2x9oFwNcgmYQy-DOHEYjwFzzyiZcJtfhpTVrDCCjzxNyH7IL9-CFwgXnYHETNsSlZbDym_nNySO_JZs0K3wFSDDRiGrXbKIUi3bOUNbWFaQo3qeC7EvuUV04sipvTsvMEMnKBMc74Reov-Qw5TaO5Z51rbVutmn5AkidVYljUfNON-OgxDZ0_656Htt-iX-9cyrXbJGUTakcs0TTmDibMyZlsJsLhTPtcGPrWn3Mf7TPMjBvqOxw Download .mp4 (1.25 MB) Help with .mp4 files Supplementary Movie 2Macrophage (green)- and nerve fiber (red) distribution in the gastric myenteric plexus.

BACKGROUND:Gastric emptying is a complex physiological process regulating the division of a meal into smaller partitions for the small intestine. Disrupted gastric emptying contributes to digestive disease, yet current measures may not reflect different mechanisms by which the process can be altered. METHODS:We have developed high temporal resolution solid and liquid gastric emptying breath tests in mice using [13 C]-octanoic acid and off axis- integrated cavity output spectroscopy (OA-ICOS). Stretched gamma variate and 2-component stretched gamma variate models fit measured breath excretion data. KEY RESULTS:These assays detect acceleration and delay using pharmacological (7.5 mg/kg atropine) or physiological (nutrients, cold exposure stress, diabetes) manipulations and remain stable over time. High temporal resolution resolved complex excretion curves with 2 components, which was more prevalent in mice with delayed gastric emptying following streptozotocin-induced diabetes. There were differences in the gastric emptying of Balb/c vs C57Bl6 mice, with slower gastric emptying and a greater occurrence of two-phase gastric emptying curves in the latter strain. Gastric emptying of C57Bl6 could be accelerated by halving the meal size, but with no effect on the occurrence of two-phase gastric emptying curves. A greater proportion of two-phase gastric emptying was induced in Balb/c mice with the administration of PYY (8-80 nmol) 60 min following meal ingestion. CONCLUSIONS AND INFERENCES:Collectively, these results demonstrate the utility of high temporal resolution gastric emptying assays. Two-phase gastric emptying is more prevalent than previously reported, likely involves intestinal feedback, but contributes little to the overall rate of gastric emptying.

BACKGROUND & AIMS:Muscularis propria macrophages lie close to cells that regulate gastrointestinal motor function, including interstitial cells of Cajal (ICC) and myenteric neurons. In animal models of diabetic gastroparesis, development of delayed gastric emptying has been associated with loss of macrophages that express cytoprotective markers and reduced networks of ICC. Mice with long-term diabetes and normal gastric emptying have macrophages that express anti-inflammatory markers and have normal gastric ICC. Mice homozygous for the osteopetrosis spontaneous mutation in the colony-stimulating factor 1 gene (Csf1op/op) do not have macrophages; when they are given streptozotocin to induce diabetes, they do not develop delayed gastric emptying. We investigated whether population of the gastric muscularis propria of diabetic Csf1op/op mice with macrophages is necessary to change gastric emptying, ICC, and myenteric neurons and investigated the macrophage-derived factors that determine whether diabetic mice do or do not develop delayed gastric emptying. METHODS:Wild-type and Csf1op/op mice were given streptozotocin to induce diabetes. Some Csf1op/op mice were given daily intraperitoneal injections of CSF1 for 7 weeks; gastric tissues were collected and cellular distributions were analyzed by immunohistochemistry. CD45+, CD11b+, F4/80+ macrophages were dissociated from gastric muscularis propria, isolated by flow cytometry and analyzed by quantitative real-time polymerase chain reaction. Cultured gastric muscularis propria from Csf1op/op mice was exposed to medium that was conditioned by culture with bone marrow-derived macrophages from wild-type mice. RESULTS:Gastric muscularis propria from Csf1op/op mice given CSF1 contained macrophages; 11 of 15 diabetic mice given CSF1 developed delayed gastric emptying and had damaged ICC. In non-diabetic Csf1op/op mice, administration of CSF1 reduced numbers of gastric myenteric neurons but did not affect the proportion of nitrergic neurons or ICC. In diabetic Csf1op/op mice given CSF1 that developed delayed gastric emptying, the proportion of nitrergic neurons was the same as in non-diabetic wild-type controls. Medium conditioned by macrophages previously exposed to oxidative injury caused damage to ICC in cultured gastric muscularis propria from Csf1op/op mice; neutralizing antibodies against IL6R or TNF prevented this damage to ICC. CD45+, CD11b+, and F4/80+ macrophages isolated from diabetic wild-type mice with delayed gastric emptying expressed higher levels of messenger RNAs encoding inflammatory markers (IL6 and inducible nitric oxide synthase) and lower levels of messenger RNAs encoding markers of anti-inflammatory cells (heme oxygenase 1, arginase 1, and FIZZ1) than macrophages isolated from diabetic mice with normal gastric emptying. CONCLUSIONS:In studies of Csf1op/op and wild-type mice with diabetes, we found delayed gastric emptying to be associated with increased production of inflammatory factors, and reduced production of anti-inflammatory factors, by macrophages, leading to loss of ICC.

In the gastrointestinal (GI) epithelium, enterochromaffin (EC) cells are enteroendocrine cells responsible for producing >90% of the body's serotonin (5-hydroxytryptamine, 5-HT). However, the molecular mechanisms of EC cell function are poorly understood. Here, we found that EC cells in mouse primary cultures fired spontaneous bursts of action potentials. We examined the repertoire of voltage-gated sodium channels (NaV) in fluorescence-sorted mouse EC cells and found that Scn3a was highly expressed. Scn3a-encoded NaV1.3 was specifically and densely expressed at the basal side of both human and mouse EC cells. Using electrophysiology, we found that EC cells expressed robust NaV1.3 currents, as determined by their biophysical and pharmacologic properties. NaV1.3 was not only critical for generating action potentials in EC cells, but it was also important for regulating 5-HT release by these cells. Therefore, EC cells use Scn3a-encoded voltage-gated sodium channel NaV1.3 for electrical excitability and 5-HT release. NaV1.3-dependent electrical excitability and its contribution to 5-HT release is a novel mechanism of EC cell function.

New Findings What is the central question of this study? The aim was to investigate the roles of extracellular chloride in electrical slow waves and resting membrane potential of mouse jejunal smooth muscle by replacing chloride with the impermeant anions gluconate and isethionate. What is the main finding and its importance? The main finding was that in smooth muscle cells, the resting Cl− conductance is low, whereas transmembrane Cl− movement in interstitial cells of Cajal (ICCs) is a major contributor to the shape of electrical slow waves. Furthermore, the data confirm that ICCs set the smooth muscle membrane potential and that altering Cl− homeostasis in ICCs can alter the smooth muscle membrane potential. Intracellular Cl− homeostasis is regulated by anion‐permeable channels and transporters and contributes to excitability of many cell types, including smooth muscle and interstitial cells of Cajal (ICCs). Our aims were to investigate the effects on electrical activity in mouse jejunal muscle strips of replacing extracellular Cl− (Cl−o) with the impermeant anions gluconate and isethionate. On reducing Cl−o, effects were observed on electrical slow waves, with small effects on smooth muscle membrane voltage (Em). Restoration of Cl− hyperpolarized smooth muscle Em proportional to the change in Cl−o concentration. Replacement of 90% of Cl−o with gluconate reversibly abolished slow waves in five of nine preparations. Slow waves were maintained in isethionate. Gluconate and isethionate substitution had similar concentration‐dependent effects on peak amplitude, frequency, width at half peak amplitude, rise time and decay time of residual slow waves. Gluconate reduced free ionized Ca2+ in Krebs solutions to 0.13 mm. In Krebs solutions containing normal Cl− and 0.13 mm free Ca2+, slow wave frequency was lower, width at half peak amplitude was smaller, and decay time was faster. The transient hyperpolarization following restoration of Cl−o was not observed in W/Wv mice, which lack pacemaker ICCs in the small intestine. We conclude that in smooth muscle cells, the resting Cl− conductance is low, whereas transmembrane Cl− movement in ICCs plays a major role in generation or propagation of slow waves. Furthermore, these data support a role for ICCs in setting smooth muscle Em and that altering Cl− homeostasis in ICCs can alter smooth muscle Em.

The production and handling of serotonin (5‐ HT ) is an important determinant of colonic motility and has been reported to be altered in gastrointestinal ( GI ) disorders such as irritable bowel syndrome ( IBS ). Recent studies suggest that the intestinal microbiota and sex of the host can influence expression of genes involved in 5‐ HT biosynthesis and signaling. While expression of genes in serotonergic pathways has been shown to be variable, it remains unclear whether genes within this pathway are coregulated. As a first step in that direction, we investigated potential correlations in relative mRNA expression of serotonergic genes, in the proximal colon isolated from male and female mice in different states of microbial association: germ‐free ( GF ), humanized (ex‐germ‐free colonized with human gut microbiota, HM ), and conventionally raised ( CR ) mice. Among the 10 pairwise comparisons conducted between five serotonergic transcripts, Tph1 , Chga , Maoa , Slc6a4 , and Htr4 , we found a strong, positive correlation between colonic expression of Slc6a4 and Htr4 across different colonization states and sexes. We also identified a positive correlation between the expression of Tph1 and Chga ; however, there were no correlations observed between any other tested pair of 5‐ HT ‐related transcripts. These data suggest that correlated expression of Slc6a4 and Htr4 likely involves coregulation of genes located on different chromosomes which modulate serotonergic activity in the gut. Further work will need to be done to understand the pathways and cell types responsible for this correlated expression, given the important role of 5‐ HT in gastrointestinal physiology.

Nutritional interventions often fail to prevent growth failure in childhood and adolescent malnutrition and the mechanisms remain unclear. Recent studies revealed altered microbiota in malnourished children and anorexia nervosa. To facilitate mechanistic studies under physiologically relevant conditions, we established a mouse model of growth failure following chronic dietary restriction and examined microbiota in relation to age, diet, body weight, and anabolic treatment.

BACKGROUND & AIMS: Gastroparesis is a complication of diabetes characterized by delayed emptying of stomach contents and accompanied by early satiety, nausea, vomiting, and pain. No safe and reliable treatments are available. Interleukin 10 (IL10) activates the M2 cytoprotective phenotype of macrophages and induces expression of heme oxygenase 1 (HO1) protein. We investigated whether IL10 administration could improve gastric emptying and reverse the associated cellular and electrical abnormalities in diabetic mice.METHODS: Nonobese diabetic mice with delayed gastric emptying were given either IL10 (0.1-1 mu g, twice/day) or vehicle (controls). Stomach tissues were isolated, and sharp microelectrode recordings were made of the electrical activity in the gastric muscle layers. Changes to interstitial cells of Cajal (ICC), reduced nicotinamide adenine dinucleotide phosphate diaphorase, and levels and distribution of HO1 protein were determined by histochemical and imaging analyses of the same tissues.RESULTS: Gastric emptying remained delayed in vehicle-treated diabetic mice but returned to normal in mice given IL10 (n = 10 mice; P < .05). In mice given IL10, normalization of gastric emptying was associated with a membrane potential difference between the proximal and distal stomach, and lower irregularity and higher frequency of slow-wave activity, particularly in the distal stomach. Levels of HO1 protein were higher in stomach tissues from mice given IL10, and ICC networks were more organized, better connected, and more evenly distributed compared with controls.CONCLUSIONS: IL10 increases gastric emptying in diabetic mice and has therapeutic potential for patients with diabetic gastroparesis. This response is associated with up-regulation of HO1 and repair of connectivity of ICC networks.

BACKGROUND & AIMS:Diabetic gastroparesis is associated with changes in interstitial cells of Cajal (ICC), neurons and smooth muscle cells in both animal models and humans. Macrophages appear to be critical to the development of cellular damage that leads to delayed gastric emptying but the mechanisms involved are not well understood. Csf1op/op (Op/Op) mice lack biologically active Csf1, resulting in the absence of Csf1-dependent tissue macrophages. The aim of this study was to use Csf1op/op mice to determine the role of macrophages in the development of delayed gastric emptying. METHODS:Animals were injected with streptozotocin to make them diabetic. Gastric emptying was determined weekly. Immunohistochemistry was used to identify macrophages and ICC networks in the gastric muscular layers. Oxidative stress was measured by serum malondialdehyde (MDA) levels. Quantitative, reverse transcription PCR was used to measure levels of mRNA. RESULTS:Csf1op/op mice had normal ICC. With onset of diabetes both Csf1op/op and wild type Csf1+/+ mice developed increased levels of oxidative stress (75.8 ± 9.1 and 41.2±13.6 nmol/mL MDA respectively). Wild type Csf1+/+ mice developed delayed gastric emptying after onset of diabetes (4/13) whereas no diabetic Csf1op/op mouse developed delayed gastric emptying (0/15, P=0.035). ICC were disrupted in diabetic wild type Csf1+/+ mice with delayed gastric emptying but remained normal in diabetic Csf1op/op mice. CONCLUSIONS:Cellular injury and development of delayed gastric emptying in diabetes requires the presence of muscle layer macrophages. Targeting macrophages may be an effective therapeutic option to prevent cellular damage and development of delayed gastric emptying in diabetes.

Background Otilonium bromide (OB) is used as a spasmolytic drug in the treatment of the functional bowel disorder irritable bowel syndrome. Although its acute effects on colonic relaxation are well-characterized, little is known about the effects of chronic administration of OB on enteric neurons, neuromuscular transmission, and interstitial cells of Cajal (ICC), key regulators of the gut function. Methods Adult Sprague-Dawley rats were treated with OB in drinking water at a dose of 2 mg/kg for 30 days. The colons of OB-treated and age-matched control rats were studied by confocal immunohistochemistry to detect immunoreactivity (IR) in myenteric plexus neurons for nitrergic and tachykininergic markers, and also by microelectrode electrophysiology. Key Results Using immunohistochemistry, chronic OB administration did not change total neuron number, assessed by anti-Hu IR, but resulted in a significant increase in NK1 receptor positive neurons, a decrease in neuronal nitric oxide synthase expressing neurons, and a reduction in volume of substance P in nerve fibers in the myenteric plexus. Chronic OB administration potentiated inhibitory and excitatory junction potentials evoked by repetitive electrical field stimulation. The various types of colonic ICC, detected by Kit IR, were not altered nor were slow waves or smooth muscle membrane potential. Conclusions & Inferences Chronic treatment with OB caused significant changes in the nitrergic and tachykinergic components of the myenteric plexus and in both inhibitory and excitatory neurotransmission in the rat colon.

H 2 S is generated by CSE in the muscle layers of the mouse colon. The aim of this study was to study the effect of H 2 S on mouse colon circular smooth muscle cells and investigate a possible link between changes in the electrical activity and the muscle tone. Result: Superfusion of NaHS (0.2 mM) had no significant effect on resting membrane potential but inhibited ongoing myoelectric complexes of muscle cells and induced a biphasic change in muscle tone: initial muscle contraction followed by relaxation. Washing out NaHS restored the ongoing myoelectric complexes but did not restore resting muscle tone suggesting that relaxation was not associated with electric activity of smooth muscle cells. NaHS reduced the frequency and amplitude of spontaneous inhibitory junction potentials (IJPs) with a time course similar to muscle contraction. Also, NaHS reduced NO-mediated slow-IJPs evoked by field stimulation suggesting H 2 S inhibits NO production. We further tested NO generation in wild type and CSE-KO mouse preparations. Both tissue levels of NO and the amplitude of evoked slow-IJPs were significantly higher in muscle layers of CSE-KO mice. Our results suggest that the action of H 2 S on colonic smooth muscle cell physiology involving brief contraction and irreversible relaxation, inhibition of myoelectrical activity, and inhibition of NO likely occurs via three independent mechanisms. The inhibition of NO contributes to the H 2 S-induced muscle contraction phase (Support: NIH DK17238).