Fig. 2 |. Bacteria activate nociceptors that release CGRP in meninges.
a, Illustration of meninges explant CGRP assays from mice infected with S. pneumoniae (3 × 107 c.f.u.) or S. agalactiae (1 × 108 c.f.u.). EIA, enzyme immunoassay. b, CGRP release induced by S. pneumoniae. n = 5 (uninfected), n = 3 (6 h), n = 5 (24 h), n = 4 (48 h). c, CGRP released from Nav1.8-DTA and control mice 24 h after S. pneumoniae injection (n = 6 per group). d, CGRP released 24 h after S. pneumoniae or S. agalactiae injection (n = 5 per group). e, Dura mater showing bacteria near CGRP+ nociceptors 24 h after S. pneumoniae (3 × 107 c.f.u.) or S. agalactiae (1 × 108 c.f.u.) infection. Scale bar, 5 μm. f, Representative fields (left) and calcium traces (centre) of trigeminal neurons responding to S. pneumoniae (4 × 107 c.f.u.), capsaicin (Caps; 1 μM) and/or KCl (40 mM). Color bar, F340/F380 ratio. Scale bar, 100 μm. Right, proportions of capsaicin-nonresponsive and capsaicin-responsive neurons responding to S. pneumoniae. n = 6 (medium, 4 × 105 c.f.u.) or n = 5 (4 × 106, 4 × 107 c.f.u.). g, CGRP concentrations in trigeminal neuron supernatants incubated with S. pneumoniae (4 × 107 c.f.u.) for 30 min. n = 5 per group. h, Representative fields (left) and calcium traces (centre) of trigeminal neurons responding to S. agalactiae (2 × 109 c.f.u.), capsaicin (1 μm) and/or KCl (40 mM). Color bar, F340/F380 ratio. Scale bar, 100 μm. Right, capsaicin-nonresponsive and capsaicin-responsive neurons responding to S. agalactiae (2 × 107 to 2 × 109 c.f.u.). n = 4 per group. i, CGRP levels from trigeminal neurons incubated for 30 min with S. agalactiae (2 × 109 c.f.u.). n = 7 (medium) or n = 10 (S. agalactiae). j, Capsaicin-nonresponsive and capsaicin-responsive neurons to WT or Δply S. pneumoniae (4 × 106 c.f.u.). n = 6 per group. k, Left, calcium traces of neurons responding to PLY (1 μg ml−1), capsaicin (1 μM) and/or KCl (40 mM). Right, proportions of capsaicin-nonresponsive and capsaicin-responsive neurons to PLY. n = 3 (0–0.01 μg ml−1) or n = 4 (0.1–1 μg ml−1). l, CGRP levels from trigeminal neurons stimulated with PLY (0.01–1 μg ml−1) for 30 min. n = 4 per group. Statistical analysis: one-way ANOVA with Tukey post-tests (b,d,f,h,j–l) or unpaired two-sided t-tests (c,g,i). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n = biologically independent samples from mouse tissues (b–d) and cells (f–l). Mean ± s.e.m. shown. Exact P values are provided in Supplementary Table 1. Illustration in a was created with BioRender.com.