Fig. 6: In vivo calcium imaging of colonic MP in response to VNS.

a Cartoon depiction of a strategy for tracing the vagus nerve from the colon. b Native fluorescence whole-map analysis of left- and right- ganglion and vagus nerves (white arrow) after colonic infection with CTB-555 in C57BL/6J mice. The yellow arrows point to infected neurons. All scale bars are 200 μm. c Schematic of in vivo imaging of mouse colon, the purple box shows a detailed view of the intestinal window after implantation and the blue shows electrode implantation for the right VNS. d Frames captured during in vivo confocal imaging of colonic myenteric neurons expressing GCaMP6s at 59, 184.5, and 311.5 seconds after 5, 10, and 15 Hz VNS. All scale bars are 50 μm. e Changes in calcium intensity (ΔF/F₀) after VNS at different frequencies in the region of interest (ganglion and neurons) in (d). Each of the three different frequencies was stimulated 3 times, with an interval of approximately 30 s between stimulation at the same frequency and 60 s between stimulation at different frequencies. ΔF/F₀ = 200% for the vertical coordinates of cells 4 and 5, and 50% for the others. f The calcium intensity of ganglion changed during 30 seconds before and after the first stimulation at 5, 10, and 15 Hz VNS. g Changes in colonic tissue movement throughout vagal stimulation. h Tissue movement and Ca²⁺ intensity of ganglion changes over a 30-second period before and after 5, 10, and 15 Hz VNS. The red lines refer to tissue movement and the blue refer to calcium intensity of ganglion. i The calcium intensity of cells changed (ΔF/F₀) after 5, 10, and 15 Hz VNS (F_{1.1, 3.2} = 0.74, p = 0.57, n = 4). j Percentages of responsive neurons per ganglion after 5, 10, and 15 Hz VNS did not change (n = 4). (k) Tissue max movement after 5, 10, and 15 Hz VNS (F_{1.0, 2.0} = 2.9, p = 0.23, n = 3). i–k *P < 0.05, RM one-way ANOVA, Tukey post hoc test, Multiple comparisons. All data are mean ± s.e.m.