Journal: bioRxiv

Article Title: Crypt and Villus Enterochromaffin Cells are Distinct Stress Sensors in the Gut

doi: 10.1101/2024.02.06.579180

Figure Lengend Snippet: (A) Basal serotonin release in crypts (left) or villi (right). gGRAB 5HT3.0 was fully activated with 20 μM serotonin at the end of recordings for normalization. (B) 10 μM A96, a TRPA1 antagonist, inhibits the tonic activity of crypt EC cells in intestinal organoids. (C) Peak normalized gGRAB 5HT3.0 response in crypts during the tonic and high K + stimulated phases. Welch’s t-test; ****p ≤ 0.0001; n = 10. (D) In each experiment, a HEK cell expressing the gGRAB 5HT2m sensor was positioned 5 μm away from a GCaMP5g-expressing organoid EC cell. The gGRAB 5HT2m and GCaMP5g signals were simultaneously measured during tonic or AITC-stimulated activity. gGRAB 5HT2m was fully activated with 500 μM serotonin at the end of recordings for normalization. (E) Peak GCaMP5g response of EC cells during tonic vs. AITC-stimulated activity. Welch’s t-test; ****p ≤ 0.0001; n = 8 and 9. (F) Peak normalized gGRAB 5HT2m response during tonic vs. AITC-stimulated activity. The dashed line indicates a normalized gGRAB 5HT2m response of 0.63, corresponding to 1 μM serotonin. Welch’s t-test; ***p ≤ 0.001; n = 8 and 9. (G) Single-cell patch-clamp recordings were performed on HEK cells positioned 5 μm away from GCaMP5g-expressing EC cells in organoids. Inward 5-HT3 currents were measured while the GCaMP5g signal was recorded. (H) Peak GCaMP5g response of EC cells during the tonic and AITC-stimulated phases. Paired t-test; ***p ≤ 0.001; n = 9. (I) Maximum inward 5-HT3 currents during tonic vs. AITC-stimulated activity. Paired t-test; ****p ≤ 0.0001; n = 9. (J) In each experiment, a HEK cell expressing GCaMP8m and 5-HT2A receptors was positioned 5 μm away from a GCaMP5g-expressing organoid EC cell. GCaMP signals from HEK and EC were simultaneously measured. (K) Peak GCaMP5g response of EC cells during the tonic and AITC-stimulated phases. Paired t-test; **p ≤ 0.01; n = 8. (L) Peak 5-HT2A-GCaMP8m biosensor response during tonic vs. AITC-stimulated activity. Paired t-test; ns: not significant; n = 8.

Article Snippet: For biosensor experiments, 200 ng pDisplay-gGRAB 5HT2m -IRES-mCherryCAAX (Addgene, #208710), 200 ng pcDNA3-5-HT2A-P2A-GCaMP8m, or 200 ng pDisplay-ATP1.0-IRES-mCherryCAAX (Addgene, #167582) was transfected to HEK293T cells in 24-well plates.

Techniques: Activity Assay, Expressing, Patch Clamp

Journal: bioRxiv

Article Title: Crypt and Villus Enterochromaffin Cells are Distinct Stress Sensors in the Gut

doi: 10.1101/2024.02.06.579180

Figure Lengend Snippet: (A) Spontaneous serotonin release in dissociated crypts from the gGRAB 5HT3.0 sensor mice. Scale bar = 5 μm. (B) In situ hybridization of Trpa1 and Tph1 in the small intestine. Tph1 is the rate-limiting enzyme in the serotonin synthesis that is expressed in EC cells in both crypts and villi. Trpa1 expression is predominantly limited to the crypts and lower villi. Bars indicate the ratio of Trpa1 -positive cells / Tph1 -positive cells. Scale bar = 30 μm. (C) AITC activates crypt but not upper villus EC cells in primary isolated crypts and villi from gGRAB 5HT3.0 sensor mice. Scale bar = 30 μm. (D) Immunohistochemical labeling of crypt and villus EC cells in Tac1-IRES-Cre;GCaMP5g-IRES-tdTomato mice. Bars indicate the ratio of tdTomato-positive cells / serotonin-positive cells. Scale bar = 100 μm. (E) (Left) A96-sensitive spontaneous TRPA1 channel opening in a crypt EC cell. The membrane potential was held at -80 mV. (F) In the same cell, A96 inhibits the spontaneous action potentials in the same EC cell. 10 μM A96 was applied as indicated above the signal. (G) Tetrodotoxin (TTX) inhibits the spontaneous GCaMP activity of EC cells. (H) Normalized dose-response curve of gGRAB 5HT2m for serotonin in the presence of 1 μM citalopram, a serotonin transporter inhibitor (EC 50 = 5.3 × 10 −7 M. Hill Slope = 0.96. n = 10). (I) GCaMP imaging of HEK cells expressing 5-HT2A receptors and GCaMP8m. The biosensor cells repeatedly responded to 1-10 nM serotonin. Scale bar = 10 μm.

Article Snippet: For biosensor experiments, 200 ng pDisplay-gGRAB 5HT2m -IRES-mCherryCAAX (Addgene, #208710), 200 ng pcDNA3-5-HT2A-P2A-GCaMP8m, or 200 ng pDisplay-ATP1.0-IRES-mCherryCAAX (Addgene, #167582) was transfected to HEK293T cells in 24-well plates.

Techniques: In Situ Hybridization, Expressing, Isolation, Immunohistochemical staining, Labeling, Membrane, Activity Assay, Imaging