Journal: Frontiers in Cellular Neuroscience

Article Title: Glucagon-Like Peptide-1 Secreting L-Cells Coupled to Sensory Nerves Translate Microbial Signals to the Host Rat Nervous System

doi: 10.3389/fncel.2020.00095

Figure Lengend Snippet: L-cells facilitate indole-evoked activation of vagal afferents. (A) The plot illustrates basolateral secretion of Glucagon-like peptide-1 (GLP-1) from colonic tissue apically exposed to indole (1 mM, 30 min) or saline (control). (B) The schematic (amended from Buckley and O’Malley, ) illustrates the ex vivo distal colonic tissue preparation. Recording is made from vagal afferents attached to the esophagus. The mucosa can be left intact, or removed to expose enteric neurons and associated afferent endings. (C) The rectified and raw (in boxes) traces show vagal nerve activity in response to stimulation of the distal colonic mucosa. Scatter plots illustrate peak frequency. Indole-evoked vagal nerve activity was attenuated by (D) exendin 3(9–39) amide [Ex-3(9–39)]. (E) Exendin-4 (Ex-4) stimulated vagal nerve firing when applied to the exposed neurons. (F) Tetrodotoxin (TTX) inhibited the indole-evoked response when it is applied to a hemisected piece of colonic tissue but not (G) colonic tissue with an intact mucosa. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Reagents [indole (1 mM, Sigma-Aldrich: cat. No. I3408), exendin 3(9–39) amide (10 μM, Tocris; cat. No. 2081), exendin-4 (10 μM, Tocris: cat. No. 1933) or tetrodotoxin (10 nM, Tocris: cat. No. 1078)] were applied to the colonic bath in the superfusate.

Techniques: Activation Assay, Saline, Control, Ex Vivo, Activity Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Glucagon-Like Peptide-1 Secreting L-Cells Coupled to Sensory Nerves Translate Microbial Signals to the Host Rat Nervous System

doi: 10.3389/fncel.2020.00095

Figure Lengend Snippet: Mucosal L-cells facilitate indole-evoked activation of submucosal neurons. (A) Indole did not affect intracellular calcium ([Ca 2+ ] i ) levels in submucosal neurons. (B) Immunofluorescence images show the GLP-1 receptor (GLP-1R) expression in colonic submucosal neurons following neuronal and mucosal exposure to indole. Arrows indicate increased expression at the neuronal cell membranes. Scalebar: 50 μm. (C) The GLP-1R agonist, exendin 4 (Ex-4) stimulated an increase in [Ca 2+ ] i , a reproducible effect that was (D) attenuated by the GLP-1R antagonist, exendin 3(9–39) amide [Ex-3(9–39)]. (E) The schematic illustrates a cross-section of the hemisected distal colon. By removing half of the mucosa, changes in intracellular calcium can be recorded from submucosal neurons loaded with a calcium indicator dye. By leaving some of the mucosa intact, the role of endocrine hormones secreted by epithelial cells can be investigated. (F) In hemisected colonic tissue, the Ex-4-evoked calcium response was enhanced by mucosal indole. (G) Immunofluorescent images show GLP-1R expression in colonic submucosal neurons following mucosal stimulation with saline, cryoprotectant, broth, L. paracasei secretions, and L. paracasei bacteria. Scalebar: 50 μm. (H) Mucosal application of L. paracasei secretions evoked a calcium response in submucosal neurons which was attenuated by the GLP-1R antagonist, exendin 3(9–39) amide [Ex-3(9–39)]. *** p < 0.001.

Article Snippet: Reagents [indole (1 mM, Sigma-Aldrich: cat. No. I3408), exendin 3(9–39) amide (10 μM, Tocris; cat. No. 2081), exendin-4 (10 μM, Tocris: cat. No. 1933) or tetrodotoxin (10 nM, Tocris: cat. No. 1078)] were applied to the colonic bath in the superfusate.

Techniques: Activation Assay, Immunofluorescence, Expressing, Saline, Bacteria

Journal: Frontiers in Cellular Neuroscience

Article Title: Glucagon-Like Peptide-1 Secreting L-Cells Coupled to Sensory Nerves Translate Microbial Signals to the Host Rat Nervous System

doi: 10.3389/fncel.2020.00095

Figure Lengend Snippet: Lactobacillus paracasei secretions stimulate host neurons. (A) The representative immunofluorescent image of a rat colonic cross-section illustrates that some L-cells expressed GLP-1Rs (arrowhead) but others did not (arrow). (B) Mucosal application of exendin-4 (Ex-4) increased vagal activity. (C) Increased vagal nerve activity evoked by L. paracasei secretions was abolished by Ex-3(9–39). * p < 0.05, ** p < 0.01. Scale bars: 50 μm.

Article Snippet: Reagents [indole (1 mM, Sigma-Aldrich: cat. No. I3408), exendin 3(9–39) amide (10 μM, Tocris; cat. No. 2081), exendin-4 (10 μM, Tocris: cat. No. 1933) or tetrodotoxin (10 nM, Tocris: cat. No. 1078)] were applied to the colonic bath in the superfusate.

Techniques: Activity Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Glucagon-Like Peptide-1 Secreting L-Cells Coupled to Sensory Nerves Translate Microbial Signals to the Host Rat Nervous System

doi: 10.3389/fncel.2020.00095

Figure Lengend Snippet: Exendin-4 stimulates calcium release from intracellular stores and influx via voltage-gated calcium channels. (A) The calcium response evoked by exendin-4 (Ex-4, 10 μM) was attenuated by thapsigargin (100 nM, n = 35) and (B) removal of extracellular calcium ( n = 17). Specific inhibitors of (C) P/Q-(ω agatoxin IVA 100 nM, n = 9) and (D) N-(ω-conotoxin GVIA, 100 nM, n = 14) type voltage gated calcium channels inhibited Ex-4-evoked calcium responses. (E) The phosphoinositide 3-kinase (PI 3-kinase) inhibitor, wortmannin (100 nM, n = 11), and (F) the STAT3 inhibitor, WP1006 (1 μM, n = 8) attenuated Ex-4-evoked calcium responses. (G) The ERK-MAPK inhibitor, PD98059 (1 μM, n = 15), had no effect. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Reagents [indole (1 mM, Sigma-Aldrich: cat. No. I3408), exendin 3(9–39) amide (10 μM, Tocris; cat. No. 2081), exendin-4 (10 μM, Tocris: cat. No. 1933) or tetrodotoxin (10 nM, Tocris: cat. No. 1078)] were applied to the colonic bath in the superfusate.

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: Glucagon-Like Peptide-1 Secreting L-Cells Coupled to Sensory Nerves Translate Microbial Signals to the Host Rat Nervous System

doi: 10.3389/fncel.2020.00095

Figure Lengend Snippet: Exendin-4 enhances colonic secretory currents. (A) The representative secretory traces from Ussing chamber studies and the associated scatter plots illustrate the secretory current evoked by the GLP-1R agonist, exendin 4 (Ex-4, 10 μM, n = 8). (B) Ex-4 does not modify the cholinergically-mediated secretory current (I SC ) evoked by carbachol (10 μM, n = 8) but enhances currents evoked by the (C) sodium channel agonist, veratridine (10 μM, n = 8) and (D) the sensory nerve stimulant, capsaicin (1 μM, n = 8). (E) Ex-4 also increases transepithelial resistance (TER) throughout an experiment (60–90 min). * p < 0.05, ** p < 0.01.

Article Snippet: Reagents [indole (1 mM, Sigma-Aldrich: cat. No. I3408), exendin 3(9–39) amide (10 μM, Tocris; cat. No. 2081), exendin-4 (10 μM, Tocris: cat. No. 1933) or tetrodotoxin (10 nM, Tocris: cat. No. 1078)] were applied to the colonic bath in the superfusate.

Techniques: