Beyder, Arthur

AbstractBackgroundGastrointestinal (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.MethodWe 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 ResultWe 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 & InferencesA 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.

AbstractBackgroundThe 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.MethodsAdvillin 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 ResultsAdvillin 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 InferencesAdvillin 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 Na V 1.5. 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 Na V 1.5. Within this set, a novel Na V 1.5 regulator, let-7f, resulted in decreased Na V 1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest Na V 1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.

Significance Mechanical forces are important for normal gastrointestinal tract function. The enterochromaffin cells in the gastrointestinal epithelium have been proposed, but not previously shown, to be specialized sensors that convert forces into serotonin release, and serotonin released from these cells is important for normal gastrointestinal secretion and motility. The findings in this study show that some enterochromaffin cells are indeed mechanosensitive, and that they use mechanosensitive Piezo2 channels to generate an ionic current that is critical for the intracellular Ca 2+ increase, serotonin release, and epithelial fluid secretion.