In vitro validation of Nes ^ER-Cre R26-STOP ^fl/fl tdTomato fate-mapping strategy to detect newborn neurons. (A) Confocal images of primary adult myenteric neuronal cultures derived from colonic muscularis of tamoxifen-treated control R26-STOP^fl/fltdTomato (Cre^–, top) and Nes^ER-CreR26-STOP^fl/fltdTomato (Cre⁺, bottom) mice, stained with Hu (neuronal somas), tdTomato (Nestin⁺ precursor–derived cells), and βIII-tubulin (nerve fibers) antibodies, and DAPI (nuclei). Scale bars, 100 μm. (B) Confocal images of primary adult myenteric neuronal cultures described in A stained with PGP9.5 (whole neurons), tdTomato (Nestin⁺ precursor–derived cells), and DAPI (nuclei). Scale bars, 100 μm. (C) Confocal images of primary adult myenteric neuronal cultures derived from colonic muscularis of naïve Nes^ER-CreR26-STOP^fl/fltdTomato (Cre⁺, top) and control SLICK-H^ER-CreR26-STOP^fl/fltdTomato (Cre⁺, bottom) mice and treated with 4-hydroxytamoxifen in vitro for 24 or 48 h, stained with PGP9.5 (whole neurons), tdTomato (Cre-targeted cells), and βIII-tubulin (nerve fibers) antibodies, and DAPI (nuclei). Scale bars, 100 μm. (D)Nes expression in mature neurons differentiated in vitro from the myenteric plexus of WT adult mice (2 wk in cell culture) and treated with LPS or vehicle as in Fig. 6 F, normalized to Actb (n = 3, n—replica wells in the same cell culture). (E)Nes expression by qPCR in CD45^–CD90⁺CD24⁺ myenteric neurons FACS-purified from water- and DSS-treated mice on day 7 after starting the DSS regimen, normalized to Actb (n = 2–3, n—independent cell sorting). All graphs show the mean ± SEM. Data are representative of at least two independent experiments with similar results. Statistical analyses: unpaired Student’s t test (D), *P ≤ 0.05.