Dataset Information

Intestinal crypt-derived enteroid coculture in presence of peristaltic longitudinal muscle myenteric plexus.

ABSTRACT: The role of enteric neurons in driving intestinal peristalsis has been known for over a century. However, in recent decades, scientists have begun to unravel additional complex interactions between this nerve plexus and other cell populations in the intestine. Investigations into these potential interactions are complicated by a paucity of tractable models of these cellular relationships. Here, we describe a novel technique for ex vivo coculture of enteroids, so called "mini-guts," in juxtaposition to the longitudinal muscle myenteric plexus (LMMP). Key to this system, we developed a LMMP culture media that: (i) allows the LMMP to maintain ex vivo peristalsis for 2 weeks along with proliferation of neurons, glia, smooth muscle and fibroblast cells, and (ii) supports the proliferation and differentiation of the intestinal stem cells into enteroids complete with epithelial enterocytes, Paneth cells, goblet cells, and enteroendocrine cells. Importantly, this technique identifies a culture condition that supports both the metabolic needs of intestinal epithelium as well as neuronal elements, demonstrating the feasibility of maintaining these two populations in a single culture system. This sets the stage for experiments to better define the regulatory interactions of these two important intestinal cell populations.

SUBMITTER: Levin DE

PROVIDER: S-EPMC7891127 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Intestinal crypt-derived enteroid coculture in presence of peristaltic longitudinal muscle myenteric plexus.

Levin Daniel E DE Mandal Arabinda A Fleming Mark A MA Bae Katherine H KH Gerry Brielle B Moore Sean R SR

Biology methods & protocols 20201223 1

The role of enteric neurons in driving intestinal peristalsis has been known for over a century. However, in recent decades, scientists have begun to unravel additional complex interactions between this nerve plexus and other cell populations in the intestine. Investigations into these potential interactions are complicated by a paucity of tractable models of these cellular relationships. Here, we describe a novel technique for <i>ex vivo</i> coculture of enteroids, so called "mini-guts," in jux ...[more]

PMID: 33628947

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Project description:BACKGROUND: Within the gut the autonomous enteric nervous system (ENS) is able to sense mechanical stimuli and to trigger gut reflex behaviour. We previously proposed a novel sensory circuit in the ENS which consists of multifunctional rapidly adapting mechanosensitive enteric neurons (RAMEN) in the guinea pig. The aim of this study was to validate this concept by studying its applicability to other species or gut regions. METHODOLOGY/PRINCIPAL FINDINGS: We deformed myenteric ganglia in the mouse small and large intestine and recorded spike discharge using voltage sensitive dye imaging. We also analysed expression of markers hitherto proposed to label mouse sensory myenteric neurons in the ileum (NF145kD) or colon (calretinin). RAMEN constituted 22% and 15% of myenteric neurons per ganglion in the ileum and colon, respectively. They encoded dynamic rather than sustained deformation. In the colon, 7% of mechanosensitive neurons fired throughout the sustained deformation, a behaviour typical for slowly adapting echanosensitive neurons (SAMEN). RAMEN and SAMEN responded directly to mechanical deformation as their response remained unchanged after synaptic blockade in low Ca(++)/high Mg(++). Activity levels of RAMEN increased with the degree of ganglion deformation. Recruitment of more RAMEN with stronger stimuli may suggest low and high threshold RAMEN. The majority of RAMEN were cholinergic but most lacked expression of NF145kD or calretinin. CONCLUSIONS/SIGNIFICANCE: We showed for the first time that fundamental properties of mechanosensitive enteric neurons, such as firing pattern, encoding of dynamic deformation, cholinergic phenotype and their proportion, are conserved across species and regions. We conclude that RAMEN are important for mechanotransduction in the ENS. They directly encode dynamic changes in force as their firing frequency is proportional to the degree of deformation of the ganglion they reside in. The additional existence of SAMEN in the colon is likely an adaptation to colonic motor patterns which consist of phasic and tonic contractions.

Dataset Information

Intestinal crypt-derived enteroid coculture in presence of peristaltic longitudinal muscle myenteric plexus.

Publications

Intestinal crypt-derived enteroid coculture in presence of peristaltic longitudinal muscle myenteric plexus.

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