Fig. 4: In vivo calcium imaging of the colonic myenteric plexus in response to SNS.

a Representative image from a Pirt-GCaMP3 mouse with GCaMP (green) expression in 18 outlined colonic myenteric neurons. b Calcium dynamics of at least 25 cells from four mice with 14 Hz SNS delivered from 30 to 45 s (vertical white dashed lines). Changes in GCaMP fluorescence are represented on a colorimetric scale. c Quantification of the percentage of GCaMP-positive cells that respond under control (ctrl, unstimulated) conditions, and with 7 Hz, 14 Hz, or 21 Hz SNS (n ≥ 3). d The percent of GCaMP-positive cells that respond under control (ctrl, unstimulated) conditions and SNS collapsed across stimulation frequency. The increase in percent response with SNS is not significant by unpaired, two-tailed t-test (n ≥ 3). e Representative image from a Pirt-GCaMP3 mouse injected with Fast Blue. GCaMP (green) expression is shown in 21 outlined colonic myenteric neurons, with Fast Blue (blue) found in 2 of the 21 cells (white/blue labels). f Changes in GCaMP fluorescence from e cells in response to 14 Hz SNS (vertical red dashed lines) in Fast-Blue-postive (blue) and -negative (black) cells. g Quantification of fraction of GCaMP-positive cells that are labeled with Fast Blue (n = 4). h Quantification of the percentage of Fast-Blue-positive cells that respond to SNS compared to all Fast-Blue-positive cells (n = 4). Circles represent individual mice, and bars indicate μ ± s.e.m. Star indicates statistically significant difference from control by Dunnett’s test for multiple comparisons after ANOVA (p < 0.05). All scale bars are 100 μm.