To calculate Ca2+ imaging data, individual cells were circled on a brightfield image and outlines overlaid onto fluorescent images using ImageJ (NIH, MA, USA). Pixel intensity was measured and analysed with custom-written scripts in RStudio (RStudio, MA, USA). Background fluorescence was subtracted from each cell, and fluorescence intensity (F) baseline corrected and normalized to the maximum fluorescence elicited during 50 mM KCl stimulation (Fpos). Maximum KCl fluorescence was denoted as 1 F/Fpos. Further analysis was confined to cells with a fluorescence increase ≥5 standard deviations above the mean baseline before 50 mM KCl application. Neurons were deemed responsive to a drug challenge if a fluorescence increase of 0.1 F/Fpos was seen in response to drug perfusion. The proportion of responsive neurons and magnitude of the fluorescence response was measured for each experiment, with peak responses calculated from averaging fluorescence values of individual neurons at each time point.
Immunocytochemistry data was obtained by first using an automatic ‘minimum error’ threshold algorithm on 8-bit images of βIII-tubulin or DAPI staining to distinguish background from objects. Binary and raw images were manually compared, and the threshold manually adjusted to ensure all regions of interest were captured. The threshold was placed at the first minimum after the major peak of the image histogram. Binary images then underwent watershed segmentation to separate distinct objects in proximity. Identified particles, positive for either βIII-tubulin or DAPI, were automatically counted using ImageJ and a ratio of βIII-tubulin-positive cells (neurons) to DAPI-positive cells (neurons and satellite cells) calculated.
In electrophysiological recordings, nerve discharge was determined by measuring the number of spikes passing a manually determined threshold twice the level of background noise (typically 60−80 μV) and binned to determine average firing frequency every 10 s. Changes in neuronal firing rates were calculated by subtracting baseline firing (averaged 3 min prior to distension or drug perfusion) from increases in nerve activity following ramp distension or capsaicin application. Peak firing to noxious mechanical distension and capsaicin application was determined respectively as the highest neuronal activity during ramp distension 5 and during the 10 min post-capsaicin application. Changes to neuronal activity were recorded with each 5 mmHg increase in pressure and used to visualize ramp profiles. Capsaicin response profiles were plotted from binned data at 30 s increments. The area under the curve (AUC) was calculated for the duration of each ramp distension (0–80 mmHg) and for the 10 min following initial capsaicin application from response profiles using GraphPad Prism 9 software.