Fig 2: Tilia tomentosa Moench (TtM) extract maintains enteric nervous system morphology. (A, D) Representative confocal microphotographs showing the distribution of HuC/D+ neurons (A and D; red), S100ß+ glial cells (A; green) and nNOS+ neurons (D; green) in longitudinal muscle-myenteric plexus (LMMP) preparations in presence of 12 µg/mL TtM pretreatment (bars = 22 µm). (B) Changes in S100ß density index in LMMP preparations in presence of 12 µg/mL TtM pretreatment. (C–E) Analysis of HuC/D+ (C) and nNOS+ (E) neurons in ileal LMMPs in presence of TtM pretreatment. Data are reported as mean ± SEM.

Fig 3: Activation of enteric glial P2Y1 receptors (P2Y1Rs) drives neuron death through a connexin-43 (Cx43)-dependent mechanism. (A) Schematic depicting how activation of neuronal P2X7 receptors (P2X7Rs) elicits Ca2+ responses in enteric glia. (B) Representative traces of in situ Ca2+ imaging of human myenteric glia. As in mice, human glia respond to the P2Y1R agonist adenosine diphosphate (ADP) and to the neuronal release of adenosine triphosphate (ATP) stimulated by the P2X7R agonist 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate triethylammonium salt (BzATP). Gray traces show responses of individual glial cells within a ganglion and the averaged response of all glia within the ganglion is overlaid in green. Traces are representative of recordings from at least 4 myenteric ganglia from the human jejunum. (C, D) In situ Ca2+ imaging of the mouse myenteric plexus demonstrates that P2Y1R agonists primarily elicit Ca2+ responses in enteric glia. (C) Representative images of Fluo-4 fluorescence in a myenteric ganglion from the mouse colon at rest (baseline) and at peak stimulation (at time = 60 seconds) with the nonhydrolyzable P2Y1R agonist adenosine 5'-[ß-thio]diphosphate trilithium salt (ADPßS). Arrows point to representative enteric glia. (D) Averaged Ca2+ responses of glia (green line) and neurons (magenta dashed line) within a myenteric ganglion from the mouse colon in response to ADPßS. Traces are representative of responses in over 5 myenteric ganglia. (E) Representative mouse myenteric ganglion showing immunoreactivity for P2Y1Rs (magenta). Enteric glia are labeled with the glial cell marker glial fibrillary acidic protein (GFAP; green) and the arrowheads highlight areas of colocalization (scale bar: 30 µM). The boxed region in the overlay image at top right is expanded in the bottom right panel to highlight a glial cell with dense P2Y1R expression. (F) Mean packing density of HuC/D-immunoreactive neurons in myenteric ganglia after in situ activation of P2Y1Rs with the agonists ADP, ADPßS and MRS2365 or activation of P2X7Rs with BzATP in the presence or absence of the Cx43 inhibitor 43Gap26 (n = 3–5 animals; **P = .01, ***P = .005, analysis of variance). (G) Mean packing density of live enteric neurons in myenteric ganglia expressed as the percentage of buffer control after in situ neuroinflammation with BzATP or ADP as quantified by calcein-AM fluorescence. Representative images show viable cells, labeled with fluorescent calcein-AM (green), in mouse myenteric ganglia from control and BzATP-treated tissues. Dead (nonfluorescent) neurons are denoted by asterisks (scale bar: 30 µM; n = 3–4 animals).

Fig 4: Oxidative stress coincides with neuron death during in vivo inflammation and is required for P2X7 receptor (P2X7R)-driven neuron death in situ. (A) Thiol oxidation measurements (ratio of fluorescently labeled oxidized/reduced glutathione) in myenteric neurons from healthy (saline) or inflamed (dinitrobenzene sulfonic acid [DNBS] colitis) animals drinking normal water (H2O) or water containing the antioxidant N-acetyl cysteine (NAC, 5 g/L) (n = 5 animals; ****P < .001, analysis of variance [ANOVA]). (B) Macroscopic damage score for animals treated in A. (C) Ganglionic fluorescence of the superoxide indicator dihydroethidium (DHE, 2 µM) in the myenteric plexus of healthy (saline) or inflamed (DNBS-colitis) mice (n = 5–6 animals; *P < .05, unpaired t test). (D) Representative myenteric ganglia from in situ preparations treated with 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate triethylammonium salt (BzATP) in the presence or absence of the antioxidant NAC. Neurons are labeled with the neuronal marker HuC/D, with dead neurons denoted by asterisks (scale bar: 30 µM). (E) Mean packing density of HuC/D-immunoreactive neurons in myenteric ganglia after in situ activation of P2X7Rs with BzATP (300 µM) in the presence of NAC (n = 4 animals; *P < .05, ANOVA).

Fig 5: Transcriptomic profiling of the adult zebrafish ENS.(A) Experimental strategy for the isolation of ENS nuclei from adult Tg(sox10:Cre;Cherry) guts and nuclear RNAseq. Five biological replicates were performed per condition. (B) Volcano plot shows mean log2 fold-change (x axis) and significance (-log10 adjusted p-value) (y axis) of genes differentially expressed in Cherry+ relative to Cherry- nuclei. Genes characteristic of the ENS are highlighted in red and are more abundant in Cherry+ nuclei, whereas genes characteristic of non-neuroectodermal lineages, such as smooth muscle (purple), interstitial cells of Cajal (green) and immune associated (blue), are more abundant in Cherry- nuclei. (C) Volcano plot (as in B) in which genes previously identified in a transcriptional characterization of larval ENS neurons (Roy-Carson et al., 2017) are shown in yellow. These include established neuronal markers, such as phox2bb, ret, elavl3, elavl4, vip, and nmu. Genes enriched in the Cherry+ nuclear population but absent from the larval ENS neuron transcriptome are shown in black. These include sox10, foxd3, sox2, plp1, the mammalian orthologues of which are expressed by mouse EGCs, tfap2a, a gene required for early NC development, col28a1b, whose mammalian orthologue is a peripheral glial marker, as well as ptprz1a, and ptprz1b, which have been identified in glioblastoma stem cells. Genes with padj <0.05 (Log10p-value<1.3) and/or log2FC < 0 are shown in grey. (D,E) Confocal images of fluorescent in situ hybridization (RNAscope) using probes for sox10 (D) and foxd3 (E) on adult Tg(sox10:Cre;Cherry) gut muscularis externa preparations immunostained for Cherry (ENS lineage) and HuC/D (ENS neurons). Signal for both sox10 and foxd3 (white arrows) corresponds to non-neuronal cells (Cherry+HuC/D-, arrows) but was absent from enteric neurons (Cherry+HuC/D+, arrowheads). (F) Immunostaining of adult Tg(sox10:Cre;Cherry) gut for Sox2 (blue), Cherry (red) and HuC/D (green). Sox2 is expressed specifically by non-neuronal ENS cells. Biological replicates: D, n = 4; E, n = 6; F, n = 5. All confocal images are max projections of short confocal stacks. 10 µm scale bars shown in merge panels.