Interstitial cells of Cajal (ICCs) generate electrical slow waves, which are required for normal gastrointestinal motility. The mechanisms for generation of normal pacemaking are not fully understood. Normal gastrointestinal contractility- and electrical slow-wave activity depend on the presence of extracellular HCO3-. Previous transcriptional analysis identified enrichment of mRNA encoding the electrogenic Na+/HCO3- cotransporter (NBCe1) gene (Slc4a4) in pacemaker myenteric ICCs in mouse small intestine. We aimed to determine the distribution of NBCe1 protein in ICCs of the mouse gastrointestinal tract and to identify the transcripts of the Slc4a4 gene in mouse and human small intestinal tunica muscularis. We determined the distribution of NBCe1 immunoreactivity (NBCe1-IR) by immunofluorescent labeling in mouse and human tissues. In mice, NBCe1-IR was restricted to Kit-positive myenteric ICCs of the stomach and small intestine and submuscular ICCs of the large intestine, that is, the slow wave generating subset of ICCs. Other subtypes of ICCs were NBCe1-negative. Quantitative real-time PCR identified >500-fold enrichment of Slc4a4-207 and Slc4a4-208 transcripts ["IP3-receptor-binding protein released by IP3" (IRBIT)-regulated isoforms] in Kit-expressing cells isolated from KitcreERT2/+, Rpl22tm1.1Psam/Sj mice and from single GFP-positive ICCs from Kittm1Rosay mice. Human jejunal tunica muscularis ICCs were also NBCe1-positive, and SLC4A4-201 and SLC4A4-204 RNAs were >300-fold enriched relative to SLC4A4-202. In summary, NBCe1 protein expressed in ICCs with electrical pacemaker function is encoded by Slc4a4 gene transcripts that generate IRBIT-regulated isoforms of NBCe1. In conclusion, Na+/HCO3- cotransport through NBCe1 contributes to the generation of pacemaker activity in subsets of ICCs.NEW & NOTEWORTHY In this study, we show that the electrogenic Na+/HCO3- cotransporter, NBCe1/Slc4a4, is expressed in subtypes of interstitial cells of Cajal (ICCs) responsible for electrical slow wave generation throughout the mouse gastrointestinal tract and is absent in other types of ICCs. The transcripts of Slc4a4 expressed in mouse ICCs and human gastrointestinal smooth muscle are the regulated isoforms. This indicates a key role for HCO3- transport in generation of gastrointestinal motility patterns.

Background and Aim Muscularis macrophages (MMs) not only mediate the innate immunity, but also functionally interact with cells important for gastrointestinal motility. The aim of this study was to determine the spatial relationship and types of contacts between the MMs and neighboring cells in the muscularis propria of human and mouse stomach, small intestine, and large intestine. Methods The distribution and morphology of MMs and their contacts with other cells were investigated by immunohistochemistry and transmission electron microscopy. Key Results Immunohistochemistry showed variable shape and number of MMs according to their location in different portions of the muscle coat. By double labeling, a close association between MMs and neighboring cells, that is, neurons, smooth muscle cells, interstitial cells of Cajal (ICCs), telocytes (TCs)/PDGFR alpha-positive cells, was seen. Electron microscopy demonstrated that in the muscle layers of both animal species, MMs have similar ultrastructural features and have specialized cell-to-cell contacts with smooth muscle cells and TCs/PDGFR alpha-positive cells but not with ICCs and enteric neurons. Conclusion & Inferences This study describes varying patterns of distribution of MMs between different regions of the gut, and reports the presence of distinct and extended cell-to-cell contacts between MMs and smooth muscle cells and between MMs and TCs/PDGFR alpha-positive cells. In contrast, MMs, although close to ICCs and nerve elements, did not make contact with them. These findings indicate specialized and variable roles for MMs in the modulation of gastrointestinal motility whose significance should be more closely investigated in normal and pathological conditions.

BACKGROUND:Normal gastrointestinal motility depends on electrical slow-wave activity generated by interstitial cells of Cajal (ICC) in the tunica muscularis of the gastrointestinal tract. A requirement for HCO3- in extracellular solutions used to record slow waves indicates a role for HCO3- transport in ICC pacemaking. The Slc4a4 gene transcript encoding the electrogenic Na+ /HCO3- cotransporter, NBCe1, is enriched in mouse small intestinal myenteric region ICC (ICC-MY) that generate slow waves. This study aimed to determine how extracellular HCO3- concentrations affect electrical activity in mouse small intestine and to determine the contribution of NBCe1 activity to these effects.METHODS:Immunohistochemistry and sharp electrode electrical recordings were used.KEY RESULTS:The NBCe1 immunoreactivity was localized to ICC-MY of the tunica muscularis. In sharp electrode electrical recordings, removal of HCO3- from extracellular solutions caused significant, reversible, depolarization of the smooth muscle and a reduction in slow-wave amplitude and frequency. In 100 mM HCO3- , the muscle hyperpolarized and slow wave amplitude and frequency increased. The effects of replacing extracellular Na+ with Li+ , an ion that does not support NBCe1 activity, were similar to, but larger than, the effects of removing HCO3- . There were no additional changes to electrical activity when HCO3- was removed from Li+ containing solutions. The Na+ /HCO3- cotransport inhibitor, S-0859 (30µM) significantly reduced the effect of removing HCO3- on electrical activity.CONCLUSIONS & INFERENCES:These studies demonstrate a major role for Na+ /HCO3- cotransport by NBCe1 in electrical activity of mouse small intestine and indicated that regulation of intracellular acid:base homeostasis contributes to generation of normal pacemaker activity in the gastrointestinal tract.

BACKGROUND:The gut is the only organ system with intrinsic neural reflexes. Intrinsic primary afferent neurons (IPANs) of the enteric nervous system initiate intrinsic reflexes, form gut-brain connections, and undergo considerable neuroplasticity to cause digestive diseases. They remain inaccessible to study in mice in the absence of a selective marker. Advillin is used as a marker for primary afferent neurons in dorsal root ganglia. The aim of this study was to test the hypothesis that advillin is expressed in IPANs of the mouse jejunum.METHODS:Advillin expression was assessed with immunohistochemistry and using transgenic mice expressing an inducible Cre recombinase under the advillin promoter were used to drive tdTomato and the genetically encoded calcium indicator GCaMP5. These mice were used to characterize the morphology and physiology of advillin-expressing enteric neurons using confocal microscopy, calcium imaging, and whole-cell patch-clamp electrophysiology.KEY RESULTS:Advillin is expressed in about 25% of myenteric neurons of the mouse jejunum, and these neurons demonstrate the requisite properties of IPANs. Functionally, they demonstrate calcium responses following mechanical stimuli of the mucosa and during antidromic action potentials. They have Dogiel type II morphology with neural processes that mostly remain within the myenteric plexus, but also project to the mucosa and express NeuN and calcitonin gene-related peptide (CGRP), but not nNOS.CONCLUSIONS AND INFERENCES:Advillin marks jejunal IPANs providing accessibility to this important neuronal population to study and model digestive disease.

Objective This study was designed to evaluate the roles of microRNAs (miRNAs) in slow transit constipation (STC). Design All human tissue samples were from the muscularis externa of the colon. Expression of 372 miRNAs was examined in a discovery cohort of four patients with STC versus three age/sex-matched controls by a quantitative PCR array. Upregulated miRNAs were examined by quantitative reverse transcription qPCR (RT-qPCR) in a validation cohort of seven patients with STC and age/sex-matched controls. The effect of a highly differentially expressed miRNA on a custom human smooth muscle cell line was examined in vitro by RT-qPCR, electrophysiology, traction force microscopy, and ex vivo by lentiviral transduction in rat muscularis externa organotypic cultures. Results The expression of 13 miRNAs was increased in STC samples. Of those miRNAs, four were predicted to target SCN5A, the gene that encodes the Na+ channel NaV1.5. The expression of SCN5A mRNA was decreased in STC samples. Let-7f significantly decreased Na+ current density in vitro in human smooth muscle cells. In rat muscularis externa organotypic cultures, overexpression of let-7f resulted in reduced frequency and amplitude of contraction. Conclusions A small group of miRNAs is upregulated in STC, and many of these miRNAs target the SCN5A-encoded Na+ channel NaV1.5. Within this set, a novel NaV1.5 regulator, let-7f, resulted in decreased NaV1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest NaV1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.

Background Gastrointestinal (GI) motility is a complex physiological process that is critical for normal GI function. Disruption of GI motility frequently occurs in GI diseases or as side effects of therapeutics. Whole gut transit measurements, like carmine red leading-edge transit, in mice form the cornerstone of in vivo preclinical GI motility studies. Method We have developed an easily achievable, labor-saving method to measure whole gut transit time in mice. This approach uses inexpensive, commercially available materials to monitor pellet production over time via high definition cameras capturing time-lapse video for offline analysis. Key Result We describe the assembly of our automated gut transit setup and validate this approach by comparing the results with loperamide to delay transit and conventional transit measurements. We demonstrate that compared to the control group, the loperamide group had slowed transit, evidenced by a decrease in total pellet production and prolonged whole gut transit time. The control group had an extended transit time compared with the results reported in the literature. Whole gut transit rates accelerated to times comparable to the literature by disrupting cages every 10-15 min to imitate the conventional approach, suggesting that disruption affects the assay and supports the use of an automated approach. Conclusion & Inferences A novel automated, inexpensive, and easily assembled whole gut transit setup is labor-saving and allows minimal disruption to animal behavior compared with the conventional approach.

BACKGROUND & AIMS:Gastric dysfunction in the elderly may cause reduced food intake, frailty, and increased mortality. The pacemaker and neuromodulator cells interstitial cells of Cajal (ICC) decline with age in humans, and their loss contributes to gastric dysfunction in progeric klotho mice hypomorphic for the anti-aging Klotho protein. The mechanisms of ICC depletion remain unclear. Klotho attenuates Wnt (wingless-type MMTV integration site) signaling. Here, we examined whether unopposed Wnt signaling could underlie aging-associated ICC loss by up-regulating transformation related protein TRP53 in ICC stem cells (ICC-SC). METHODS:Mice aged 1-107 weeks, klotho mice, APCΔ468 mice with overactive Wnt signaling, mouse ICC-SC, and human gastric smooth muscles were studied by RNA sequencing, reverse transcription-polymerase chain reaction, immunoblots, immunofluorescence, histochemistry, flow cytometry, and methyltetrazolium, ethynyl/bromodeoxyuridine incorporation, and ex-vivo gastric compliance assays. Cells were manipulated pharmacologically and by gene overexpression and RNA interference. RESULTS:The klotho and aged mice showed similar ICC loss and impaired gastric compliance. ICC-SC decline preceded ICC depletion. Canonical Wnt signaling and TRP53 increased in gastric muscles of klotho and aged mice and middle-aged humans. Overstimulated canonical Wnt signaling increased DNA damage response and TRP53 and reduced ICC-SC self-renewal and gastric ICC. TRP53 induction persistently inhibited G1/S and G2/M cell cycle phase transitions without activating apoptosis, autophagy, cellular quiescence, or canonical markers/mediators of senescence. G1/S block reflected increased cyclin-dependent kinase inhibitor 1B and reduced cyclin D1 from reduced extracellular signal-regulated kinase activity. CONCLUSIONS:Increased Wnt signaling causes age-related ICC loss by up-regulating TRP53, which induces persistent ICC-SC cell cycle arrest without up-regulating canonical senescence markers.

Figure 2. Effect of anti-CGRP F(ab')2 on hyperalgesia in the alcohol-induced CP rats examined by VFF test (A) and pancreatic electrode stimulation (B) .The tests were conducted before (left) and after (right) treatment.Data were presented as mean ± SEM (n = 6-7).*: significantly different from vehicle / Saline group; #: significantly different from Alcohol-CP / Isotype F(ab')2 group.

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.

Interstitial cells of Cajal (ICC) generate electrical pacemaker activity in the gastrointestinal (GI) tract known as slow waves, which regulate GI motility. ICC express both the Kit receptor tyrosine kinase protein and a Ca 2+ -activated Cl - -channel, encoded by the anoctamin1 (Ano1) protein, which is an essential contributor to the Ca 2+ cycling of ICC and slow wave pacemaking. Recent dye-loading imaging studies have demonstrated Ca 2+ transients in ICC in isolated tissue preparations. The main aim of this study was to develop a method that allows Ca 2+ transients to be registered to structural ICC network data. Confocal image stacks of ICC labeled for Kit or Ano1 and Ca 2+ recording data were processed using a thresholding protocol. The Ca 2+ transients were then registered to the ICC structural network. First, a general idea of the placement was found by mapping the field-of-view of the Ca 2+ transient data to the distorted tissue that contained the ICC network image. The errors in the registration were then corrected for by warping the internal Ca 2+ transient field according to the structural network. In data sets from tissues with induced, targeted knockdown of Ano1 expression in a subset of ICC, agreement between the Ca 2+ transient data and structural network was 68 ± 10%. This level of agreement allowed selective extraction of Ca 2+ data from Ano1-positive (Ano1+) and Ano1-negative (Ano1-) ICC. In the future, this technique will allow investigation into the functional properties of ICC in relation to the level of knockdown of specific ICC associated proteins.

NBCe1 is an electrogenic Na + bicarbonate cotransporter expressed as three isoforms. NBCe1A is mainly found in the kidney (100% activity), NBCe1B is a general isoform (~25% activity), and NBCe1C is found in the nervous system (20% activity). This project was to characterize mice with loss of NBCe1 in the kidney and urogenital tract and to test isoform activity ± IRBIT. Previous work has shown that NBCe1‐B is activated when IRBIT is present and interacts with the N‐terminus (Nt). Even though NBCe1C shares this Nt, the effects of IRBIT on human NBCe1C are unknown. Interstitial Cells of Cajal (ICC)pacemaker cells in the gastrointestinal tract ( c‐kit + ) have NBCe1C, IRBIT and require bicarbonate for normal gut activity. Developmentally similar ICC cells are found in the urogenital tract; however, NBCe1C locations within the kidney and urogenital tract are unknown. Using c‐kit ‐copGFP and NBCe1A‐knockout mice, kidneys, ureters, prostate, bladder, and urethra were dissected and analyzed via direct imaging, sectioning, and immunofluorescence. Oocytes injected with NBCe1C ± IRBIT were voltage clamped to determine the cotransporter's activity. “Strings” of c‐kit + ‐cells were found in the copGFP & nbce1A kidneys. In nbce1 (+/−) ‐(Het) mice (Figure), prostate ducts appeared enlarged, and in nbce1A − / − kidney, c‐kit protein was broadly expressed, showing cluster colocalization with NBCe1C. NBCe1C is not obviously expressed in WT or copGFP mice. Electrophysiology data showed that IRBIT activates NBCe1C by ~10‐fold. As c‐kit is also a marker of stem cells, NBCe1C/c‐kit association and c‐kit increase in nbce1A − / − mice, could suggest that NBCe1A loss elicits kidney repair itself (proliferation) and causes expression of other NBCe1‐isoforms. Support or Funding Information R25‐DK101405, R01‐DK057061, U54‐DK100227 (Mayo) and U54‐DK104310 (UW‐Madison) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Postsurgical gastric dysfunction is common, but the mechanisms are varied and poorly understood. The pylorus normally acts as an electrical barrier isolating gastric and intestinal slow waves. In this report, we present an aberrant electrical conduction pathway arising between the stomach and small intestine, following pyloric excision and surgical anastomosis, as a novel disease mechanism. A patient was referred with postsurgical gastroparesis following antrectomy, gastroduodenostomy, and vagotomy for peptic ulceration. Scintigraphy confirmed markedly abnormal 4-h gastric retention. Symptoms included nausea, vomiting, postprandial distress, and reflux. Intraoperative, high-resolution electrical mapping was performed across the anastomosis immediately before revision gastrectomy, and the resected anastomosis underwent immunohistochemistry for interstitial cells of Cajal. Mapping revealed continuous, stable abnormal retrograde slow-wave propagation through the anastomosis, with slow conduction occurring at the scar (4.0 ± 0.1 cycles/min; 2.5 ± 0.6 mm/s; 0.26 ± 0.15 mV). Stable abnormal retrograde propagation continued into the gastric corpus with tachygastria (3.9 ± 0.2 cycles/min; 1.6 ± 0.5 mm/s; 0.19 ± 0.12 mV). Histology confirmed ingrowth of atypical ICC through the scar, defining an aberrant pathway enabling transanastomotic electrical conduction. In conclusion, a “gastrointestinal aberrant pathway” is presented as a novel proposed cause of postsurgical gastric dysfunction. The importance of aberrant anastomotic conduction in acute and long-term surgical recovery warrants further investigation. NEW & NOTEWORTHY High-resolution gastric electrical mapping was performed during revisional surgery in a patient with severe gastric dysfunction following antrectomy and gastroduodenostomy. The results revealed continuous propagation of slow waves from the duodenum to the stomach, through the old anastomotic scar, and resulting in retrograde-propagating tachygastria. Histology showed atypical interstitial cells of Cajal growth through the anastomotic scar. Based on these results, we propose a “gastrointestinal aberrant pathway” as a mechanism for postsurgical gastric dysfunction.