Fig. 6.

Mechanosensitive EC cell 5-HT release depends on Piezo2. (A) 5-HT biosensor experiment showing overlaid DIC/tdTomato images with an EC cell, and (B) GCaMP5 in both the EC and 5-HT biosensor cell (lower part of image). EC cell mechanical stimulation by force probe results in (C) Ca²⁺ increase in EC cell, and (D) later in 5-HT biosensor. (E) Representative traces of Ca²⁺ responses (ΔF/F₀) during EC cell mechanical stimulation, and F, resulting 5-HT biosensor activity in a control experiment (black), with 0.1 µM ondansetron (red), 10 µM D-GsMTx4 (green), Piezo2 siRNA (brown), and NT siRNA (blue). Vertical lines represent stimulation of EC cell (dashed) and initiation of 5-HT biosensor cell response (dotted). (G) Individual (NeuroD1⁺ cells triangles, 5-HT biosensors circles) and mean ± SEM (bars) Ca²⁺ responses (ΔF/F₀) of EC cell mechanical stimulation and (H) resulting 5-HT biosensor cell activity in controls (EC cell: 3.2 ± 0.5, n = 22 and 5-HT biosensor: 2.7 ± 0.6, n = 22), with ondansetron (EC cell: 3.9 ± 0.5, n = 5 and 5-HT biosensor: 0.5 ± 0.1, n = 5), with D-GsMTx4 (EC cell: 0.1 ± 0.03, n = 6, and 5-HT biosensor: 0.19 ± 0.09, n = 6), with Piezo2 siRNA (EC cell: 0.6 ± 0.2, n = 13, and 5-HT biosensor: 0.7 ± 0.2, n = 13), and with NT siRNA (EC cell: 2.9 ± 0.4, n = 15, and 5-HT biosensor: 3.3 ± 0.7, n = 15) (*P < 0.05 when comparing D-GsMTx4 to control, and Piezo2 siRNA to NT siRNA for EC cell and biosensor cell, and when comparing ondansetron and D-GsMTx4 to control for biosensor cell only, by unpaired t test).