162 Potentiation of Descending Inhibition to Colonic Circular Smooth Muscle of Mice and Humans by Chronic Stress and Stress Hormones: A Mechanism for Stress-Induced Diarrhea?

Background: A population of multipotent stem cells capable of differentiating into neurons and glia has been isolated from adult intestine in humans and rodents. While they may provide a pool of stem cells for neurogenesis in the enteric nervous system (ENS), such a function has been difficult to demonstrate in vivo. Here we introduce a novel approach to study neurogenesis in the ENS using an ex vivo system. Methods: Using organotypic cultures of longitudinal muscle and myenteric plexus (LMMP) from Wnt1-cre;tdTomato (tdT) we performed EdU uptake assays at different time points and EdU incubations. To evaluate stem cell properties, LMMP treated with EdU was digested and flow cytometry performed with antibodies to nestin, CD49b, p75, and GFAP. We also performed a clonogenic sphereforming assay in which single cells were seeded into microwells to assess sphere forming capacity and retention of EdU signal. To evaluate for neurogenesis, LMMP treated with EdU was analyzed by flow cytometry and immunofluorescence with antibodies to neuronal and glial markers including Tuj, PGP9.5, nNOS, VIP, GFAP, and S100B. Protein expression for PTEN, pPTEN, Akt, and pAkt was analyzed by western blot and RT-PCR. Finally, organotypic cultures were treated with inhibitors BpV(phen) and LY294002, and EdU uptake assays were performed. Results: Culturing LMMP with EdU for 48h resulted in EdU uptake in 78% of neural crest cells (tdT+), indicating that the enteric nervous system has tremendous replicative capacity. Administering pulses of EdU at different time points, we found that proliferation occurred after an initial lag phase that lasted slightly over 24h. While undetectable at baseline and 24h, EdU uptake reached nearly 45% of tdT+ cells by 72h (p ,.001). Flow cytometry revealed that .90% of EdU+ neural crest cells expressed enteric neural stem cell markers nestin, CD49b, p75 and GFAP. Tracking single cells we found that EdU+ cells are capable of forming secondary NLBs suggestive of stem cell-like properties. Under differentiating conditions (withdrawal of growth factors), EdU uptake was detected in cells expressing Tuj, PGP9.5, nNOS, VIP, GFAP and S100B. This was supported with flow cytometry where .50% of cells positive for neuronal markers Tuj or PGP9.5 demonstrated EdU uptake (also seen for glial markers GFAP and S100B). Proliferation appears to be dependent on the PTEN/PI3K/Akt pathway with inhibition of PTEN (50% decrease in PTEN expression and 80-fold increase in pPTEN:PTEN (inactivation)) corresponding to activation of Akt (4-fold increase in pAkt:Akt) by 24h. Treatment of LMMP with PI3K inhibitor, LY294002, caused over 95% reduction (p,.01) and PTEN inhibitor, BpV(phen), caused 3fold increase (p,.001) in EdU uptake. Conclusion: This data suggest that the ENS is capable of neurogenesis in a PTEN dependent manner.