Sensitization of TRPV1 receptors by TNFα.

<p>A, Representative traces of currents induced by application of capsaicin (1 µM, 2 s) to WT or R192Q KI neurons in control conditions or after 4 h application of TNFα. B, Histograms show average peak amplitudes of TRPV1-mediated currents (WT control, n = 29; WT TNFα 50 ng/mL n = 19, WT TNFα 100 ng/mL, n = 10; KI control, n = 26; KI TNFα 50 ng/mL, n = 23,, KI TNFα 100 ng/mL n = 9); * = p<0.05, ** = p<0.002, *** = p<0.001.</p

Role of CGRP in KI P2X3 currents.

<p>A, Representative traces of currents induced by application of α,β-meATP (10 µM, 2 s) to WT or R192Q KI neurons in control conditions or after 2 h CGRP (1 µM) application. B, Histograms show average peak amplitudes of P2X3 receptor-mediated currents (WT control, n = 11; WT CGRP, n = 9; KI control, n = 17; KI CGRP, n = 18), * = p<0.05. C, Representative examples of currents induced by application of α,β-meATP (10 µM, 2 s) to WT or R192Q KI neurons in control conditions or after overnight application of the CGRP antagonist CGRP_8–37 (1 µM). D, Histograms show average peak amplitudes of P2X3 receptor-mediated currents (WT control, n = 23; WT CGRP_8–37, n = 23; KI control, n = 29; KI CGRP_8–37, n = 30); * = p<0.05.</p

Number of BDNF expressing neurons in KI culture and effect of BDNF deprivation.

<p>A, Examples of β-tubulin positive neurons expressing BDNF in WT or KI cultures. Left panel (green) shows BDNF expression and right panel (red) shows β-tubulin staining of the same neurons. B, Histograms quantifying % of neurons expressing BDNF: KI cultures showed significantly higher number of BDNF positive neurons. N = 4 independent experiments (8 mice), p<0.05. C, Representative traces of currents induced by application of α,β-meATP (10 µM, 2 s) to WT or R192Q KI neurons in control conditions or after overnight application of anti-BDNF antibody. D, Histograms show average peak amplitudes of P2X3 receptor-mediated currents (WT control, n = 9; WT anti-BDNF, n = 10; KI control, n = 32; KI anti-BDNF, n = 38); * = p<0.05.</p

Effect of TNFα on P2X3 receptor activity and co-expression of its TNFR2 receptors.

<p>A, Examples of TNFR2 and P2X3 co-exexpression in (wildtype) WT and R192Q (knockin) KI neurons. Left panel shows P2X3 expression (green), and right panel shows TNFR2 staining (red). B, Histograms quantifying % of cells co-expressing TNFR2 and P2X3: both WT and KI cultures show similar TNFR2 and P2X3 co-expression. N = 3 independent experiments (6 mice). C, Representative traces of currents induced by application of α,β-meATP (10 µM, 2 s) to WT or R192Q KI neurons in control conditions or after 4 h TNFα application. D, Histograms show average peak amplitudes of P2X3 receptor-mediated currents: WT control (open bar), n = 30; WT TNFα (stippled bar), n = 38; KI control (grey bar), n = 34; KI TNFα (stippled gray bar), n = 34; ** = p<0.006; * = p<0.05.</p

Expression of inflammatory mediators in WT and R192Q KI trigeminal ganglia under basal conditions.

<p><i>A</i>, Histograms quantify IL1β, IL6, IL10 and TNFα cytokine protein levels from WT or R192Q KI ganglia; <i>n</i> = 4 WT and 4 KI mice; data were normalized on total protein content, and represented as fraction of WT. * <i>p</i><0.001. <i>B</i>, Real-time RT-PCR experiments quantify IL1β, IL6, IL10 and TNFα mRNA levels in WT and R192Q KI trigeminal ganglia. PCR data were normalized with respect to corresponding GAPDH and β-Tubulin housekeeping gene expression and expressed as fraction of WT; <i>n</i> = 4 WT and 4 KI mice; * <i>p</i><0.05. <i>C</i>, Real-time RT-PCR experiments quantify MCP-1 mRNA levels in WT and R192Q KI trigeminal ganglia (expressed as in <i>B</i>); <i>n</i> = 4 WT and 4 KI mice; * <i>p</i><0.05. <i>D</i>, Representative western blot experiment of WT or R192Q KI trigeminal ganglia extracts immuno-probed with anti-MCP-1 antibodies. Actin levels were used as loading control. Histograms quantify the differences. <i>n</i> = 3; * <i>p</i><0.05.</p

LPS evoked acute TNFα expression in R192Q KI ganglia.

<p><i>A</i>, Representative confocal microscopy images of WT (top row) or KI (bottom row) trigeminal ganglion sections immunostained for Iba1 (red) or TNFα(green) after saline (left) or LPS injection (i.p., 5 h; right). LPS evokes TNFα expression in WT and KI after injection. Scale bar: 20 µm. <i>B, C,</i> Histograms quantify the occurrence of Iba1 signal (<i>B</i>) and Iba1-TNFα co-localisation (<i>C</i>) in different V1, V2 or V3 trigeminal ganglion regions from WT or KI mice, after saline or LPS injection (i.p., 5 h). ROI: 370×370 µm. <i>n</i> = 3 WT and 3 R192Q KI mice; * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001. <i>D</i>, Histograms quantify changes in TNFα mRNA fold increase in WT or R192Q KI ganglia following LPS-injection (i.p., 1 h). Data are expressed as fold increase with respect to saline-injected mice samples; <i>n</i> = 3 WT and 3 KI mice; * <i>p</i><0.05. <i>E</i>, TNFα protein levels in whole ganglia (pg cytokine/mg protein content) from LPS-injected (i.p. 5 h) WT or R192Q KI mice, expressed as fold increase with respect to saline-injected mice. <i>n</i> = 3 WT and 3 KI mice; * <i>p</i><0.05.</p

TNFα expression in WT and KI ganglia.

<p><i>A</i>, Representative confocal microscopy images of WT (top row) or R192Q KI (bottom row) trigeminal ganglion sections immunostained for Iba1 (red) or TNFα(green) in basal condition. Pseudocolor images showing areas of high (yellow) and low (blue) Iba1-TNFα expressing cell co-localization. Color scale was also included. Note TNFα immunostaining detected as spots along perimembrane regions. The larger magnification insets show immunostaining of Iba1-TNFα signal (yellow) in KI rather than WT. Scale bar: 30 µm, for large images; Scale bar: 10 µm for larger magnification insets. <i>B</i>, Histograms quantify the percentage of TNFα immunoreactivity over the total of Iba1 expressing cells in different V1, V2 or V3 trigeminal regions (ROI: 370×370 µm). <i>n</i> = 4 WT and 4 R192Q KI mice; * <i>p</i><0.05. Data are expressed as mean ± S.D.</p

Different morphology of Iba1 immunoreactive cells in WT and R192Q KI trigeminal ganglia.

<p><i>A</i>, Examples of 3D reconstruction of Iba1-positive cells (from confocal Z-stacks, each 0.5 µm-tick) from WT or R192Q KI ganglia. Note large branching of WT cell vs compact, process-free KI cell morphology. Scale bar: 5 µm. <i>B</i>, Histograms quantify average volume (µm³) of Iba1-positive cells in WT and R192Q KI ganglia, obtained from voxel analysis of 3D images. Data were collected from three independent experiments with a total of 83 cells for WT and 70 cells for R192Q KI; * <i>p</i><0.001. <i>C</i>, Histograms quantify the average volume of Iba1-positive cells from different neurons or fibers enriched areas of WT and R192Q KI ganglia; <i>n</i> = 20–50 cells (3 WT and 3 KI mice). * <i>p</i><0.05; ** <i>p</i><0.01.</p

Characterization of Iba1-positive cells in trigeminal ganglia from WT and R192Q KI mice.

<p><i>A, C, E</i>, Representative confocal images of WT or R192Q KI trigeminal ganglion sections (from V3 region) immunostained for Iba1 (red) and CD11b (<i>A</i>), ED1 (<i>C</i>), or F4/80 (<i>E</i>) in green. Nuclei were labeled with DAPI (blue). Scale bar: 40 µm. Insets represent larger magnification of immunoreactive Iba1 cells. Scale bar: 7 µm. <i>B, D, F,</i> Histograms quantify the percentage of occurrence of Iba1 signal with CD11b (<i>B</i>), ED1 (<i>D</i>) or F4/80 (<i>F</i>) in different WT and KI ganglion regions (V1, V2 or V3). Expression of different markers was quantified in Iba1 positive cells only. <i>n</i> = 3 WT and 3 KI mice; * <i>p</i><0.05; ** <i>p</i><0.01. Data are expressed as mean ± S.D.</p

Real time PCR primers.

<p>Real time PCR primers.</p