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Characterization of fetal and postnatal enteric neuronal cell lines with improvement in intestinal neural function.

ABSTRACT: BACKGROUND & AIMS:The isolation and culture of primary enteric neurons is a difficult process and yields a small number of neurons. We developed fetal and postnatal enteric neuronal cell lines using H-2K(b)-tsA58 transgenic mice (immortomice) that have a temperature-sensitive mutation of the SV40 large tumor antigen gene under the control of an interferon gamma-inducible H-2K(b) promoter element. METHODS:Enteric neuronal precursors were isolated from the intestines of E13-mouse fetuses and second day postnatal mice using magnetic immunoselection with a p75NTR antibody. The cells were maintained at the permissive temperature, 33 degrees C, and interferon-gamma for 24 or 48 hours, and then transferred to 39 degrees C in the presence of glial cell line-derived neurotrophic factor for 7 days for further differentiation. Neuronal markers were assessed by reverse-transcription polymerase chain reaction, Western blot, and immunocytochemistry. Neuronal function was assessed by transplanting these cells into the colons of Piebald or nNOS(-/-) mice. RESULTS:Expression analysis of cells showed the presence of neuronal markers peripherin, PGP9.5, HuD, tau, synaptic marker synaptophysin, characteristic receptors of enteric neurons, Ret, and 5-hydroxytryptamine-receptor subtypes at 33 degrees C and 39 degrees C. Nestin, S-100beta, and alpha-smooth muscle actin were expressed minimally at 39 degrees C. Glial cell line-derived neurotrophic factor resulted in increased phosphorylation of Akt in these cells, similar to primary enteric neurons. Transplantation of cells into the piebald or nNOS(-/-) mice colon improved colonic motility. CONCLUSIONS:We have developed novel enteric neuronal cell lines that have neuronal characteristics similar to primary enteric neurons. These cells can help us in understanding newer therapeutic options for Hirschsprung's disease.

SUBMITTER: Anitha M

PROVIDER: S-EPMC2612783 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Characterization of fetal and postnatal enteric neuronal cell lines with improvement in intestinal neural function.

Anitha Mallappa M Joseph Irene I Ding Xiaokun X Torre Enrique R ER Sawchuk Michael A MA Mwangi Simon S Hochman Shawn S Sitaraman Shanthi V SV Anania Frank F Srinivasan Shanthi S

Gastroenterology 20080214 5

<h4>Background & aims</h4>The isolation and culture of primary enteric neurons is a difficult process and yields a small number of neurons. We developed fetal and postnatal enteric neuronal cell lines using H-2K(b)-tsA58 transgenic mice (immortomice) that have a temperature-sensitive mutation of the SV40 large tumor antigen gene under the control of an interferon gamma-inducible H-2K(b) promoter element.<h4>Methods</h4>Enteric neuronal precursors were isolated from the intestines of E13-mouse fe ...[more]

PMID: 18471518

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Project description:The enteric nervous system (ENS) regulates the motor, secretory and defensive functions of the gastrointestinal tract. Enteric neurons integrate mechanical and chemical inputs from the gut lumen to generate complex motor outputs. How intact enteric neural circuits respond to changes in the gut lumen is not well understood. We recorded intracellular calcium in live-cell confocal recordings in neurons from intact segments of mouse intestine in order to investigate neuronal response to luminal mechanical and chemical stimuli. Wnt1-, ChAT- and Calb1-GCaMP6 mice were used to record neurons from the jejunum and colon. We measured neuronal calcium response to KCl (75 mM), veratridine (10 μM), 1,1-dimethyl-4-phenylpiperazinium (DMPP; 100 μM) or luminal nutrients (Ensure®), in the presence or absence of intraluminal distension. In the jejunum and colon, distension generated by the presence of luminal content (chyme and faecal pellets, respectively) renders the underlying enteric circuit unresponsive to depolarizing stimuli. In the distal colon, high levels of distension inhibit neuronal response to KCl, while intermediate levels of distension reorganize Ca2+ response in circumferentially propagating slow waves. Mechanosensitive channel inhibition suppresses distension-induced Ca2+ elevations, and calcium-activated potassium channel inhibition restores neuronal response to KCl, but not DMPP in the distended colon. In the jejunum, distension prevents a previously unknown tetrodotoxin-resistant neuronal response to luminal nutrient stimulation. Our results demonstrate that intestinal distension regulates the excitability of ENS circuits via mechanosensitive channels. Physiological levels of distension locally silence or synchronize neurons, dynamically regulating the excitability of enteric neural circuits based on the content of the intestinal lumen. KEY POINTS: How the enteric nervous system of the gastrointestinal tract responds to luminal distension remains to be fully elucidated. Here it is shown that intestinal distension modifies intracellular calcium levels in the underlying enteric neuronal network, locally and reversibly silencing neurons in the distended regions. In the distal colon, luminal distension is integrated by specific mechanosensitive channels and coordinates the dynamics of neuronal activation within the enteric network. In the jejunum, distension suppresses the neuronal calcium responses induced by luminal nutrients. Physiological levels of distension dynamically regulate the excitability of enteric neuronal circuits.

Dataset Information

Characterization of fetal and postnatal enteric neuronal cell lines with improvement in intestinal neural function.

Publications

Characterization of fetal and postnatal enteric neuronal cell lines with improvement in intestinal neural function.

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